Polychelating agents for image and spectral enhancement (and spectral shift)

ABSTRACT

The present invention includes an image-enhancing agent comprising a biodegradable, water-soluble polymer, synthetic or naturally derived and having repeating hydrophilic monomeric units with amino or hydroxyl groups. This agent also includes chelating agents comprising functional groups bound to an amino or hydroxyl group of the monomeric units. These chelating agents have a formation constant for divalent or trivalent metal cations of at least about 10 8  at physiological temperature and pH. This image-enhancing agent is biodegradable to intermediary metabolites, excretable chelates, oligomers, monomers or combinations thereof of low toxicity. These image-enhancing agents may further comprise a paramagnetic metal ion for enhancement of the image arising from induced magnetic resonance signals. Images resulting from scanning of gamma particle emissions may be enhanced when the image-enhancing agent of the present invention comprises radioisotopic metal ions emitting gamma particles. The physical conversion of these image-enhancing agents into microspheres (or, less optimally, microaggregates) allows further internal directioning of the image-enhancing agents to organs with phagocytic capabilities. Dextran is a preferred polymer; DTPA and gadolinium are respectively preferred chelating agents and paramagnetic metal ions.

This is a divisional of copending application Ser. No. 07/086,692 filedon Aug. 7, 1987 now abandoned, and international applicationPCT/US86/02479 filed on Nov. 18, 1986 and which designated the U.S.which is a continuation-in-part of pending U.S. patent application Ser.No. 799,757 filed Nov. 18, 1985, now abandoned and which is expresslyincorporated by reference herein.

The present invention relates to image-enhancing agents, contrast agentsor spectral shift agents to enhance tissue or organ images or nuclearspectra obtained from live animals with ultrasound imaging radioisotopescanning or NMR imaging or spectroscopy.

The imaging of internal structures and organs of live animals has beenan important aspect of medicine since the advent of X-ray usage for thispurpose. Among the techniques more recently developed for such imagingare those involving scanning for emission of particles from aninternally located radioisotope. Such radioisotopes preferably emitgamma particles and are generally isotopes of metallic elements. Oneproblem common to the diagnostic usage of such gamma particle-emittingradioisotopes concerns the localization of these materials at sites ofparticular interest rather than to have them randomly dispersed orrapidly excreted, by the kidney, for example. Another problem of suchradioisotope mediated imaging concerns optimizing the circulatinghalf-life of radioisotopes, for example, by preventing or accentuatingtheir binding to serum proteins (e.g., albumin), or by prior conjugation(complexation) to polymeric carriers or receptor-binding substances.

A second class of internal body imaging which is undergoing a rapidgrowth in clinical use is ultrasound imaging. This is based on thedetection of differences in the internal velocity (reflectivity) ofdirected, high-frequency sound waves. Differences in image brightnessare produced at the interfaces between tissues with different nativedensities and ultrasound reflectivities. A present clinical problem isthe difficulty of visualizing lesions in the stomach, small and largebowel, bladder, and cavities of the female reproductive tract, due tosimilarities of ultrasound velocity between these organs of interest andimmediately adjacent tissues. Diagnostic introduction of a dense,nonradioactive metal element or ion at sufficient concentrations canconfer the significant differences in ultrasound reflectivity which arerequired to visualize otherwise undetectable tumors and inflammatorylesions.

NMR intensity and relaxation images have been shown in recent years toprovide a third important method of imaging internal structures andorgans of live animals. Clinical magnetic resonance Imaging (MRI) is arapidly growing, new form of brain and body imaging. Low-field (proton)MRI detects chemical parameters in the immediate environment around theprotons of body tissues (predominantly water protons because of theirrelative abundance). Changes in these parameters occur very early indisease and are independent of physical densities detected by ionizingradiation. In the brain and central nervous system, MRI has alloweddetection of tumors at an earlier clinical stage and with fewer imagingartifacts than is possible with computerized axial tomography (CAT)(Rungs et al., (1983) Am. J. Radiol V 141, p 1209). Under optimalconditions, image resolution is in the submillimeter size range.

Seven factors make it important to develop nontoxic MRI image-enhancingagents analogous to those available for CAT. 1. They increase thespecificity of MRI diagnosis. 2. Smaller lesions can be identifiedearlier. 3. Image-enhancing agents enhance tumor masses differently thansurrounding edema fluid or abscesses. This allows the extent andinvasion of tumors to be defined more precisely. Lesions withinfiltrative-type growth (e.g., certain metastatic carcinomas andglioblastomas) will require contrast agents for demarcation betweentumor and edema fluid (Felix et al. (1985) Proc. Soc. Mag. Res. Med. V2, p 831). 4. Image-enhancing agents improve the distinction betweenrecurrent tumor and fibrous tissue resulting from surgery and radiation.5. Image-enhancing agents can decrease the time required per scan andpotentially decrease the number of scans required per procedure. Thisincreases the volume of procedures and decreases their expense. 6. Bodyimaging has a significantly lower resolution (typically 0.5-1.0 cm) andsensitivity (decreased signal-to-noise ratio) than brain imaging (Wesbeyet al. (1983) Radiology V 149, p 175). These differences result from theGreater inhomogeneity of the magnetic field; the larger radiofrequencycoil; unequal phase-pulsing of deep versus shallow nuclei; and motionartefacts produced by respiration, cardiac systole, gastrointestinalperistalsis, and voluntary muscle movement; and 7. Advanced (polymericand microsphere) forms of contrast agents (see below) appear to berequired for the optimal acquisition and interpretation of blood-flowand tissue-perfusion images and related spectral (phase) information.

The discrete intensities of a two-dimensional, Fourrier-transformedimage are described by the following general equation (for spin-echopulse sequences):

    Intensity=N(H)·f(v)·exp(-TE/T2)·(1-exp(TE-TR/T1 ), where:

N(H)=number of protons in the discrete tissue volume (spin density);

f(v)=a function of proton velocity and the fraction of protons which aremoving (e.g., due to following blood);

TE=time between the radio frequency (rf) pulse and the detection ofsignal (spin-echo);

TR=the interval between repetition of the rf pulse.

T1=the time interval associated with the rate of proton energy transferto the surrounding chemical environment (spin-lattice relaxation);

T2=the time interval associated with the rate of proton energy transfer,one to other (spin-spin relaxation).

The T1 and T2 times have reciprocal effects on image intensity.Intensity is increased by either shortening the T1 or lengthening theT2. Tissue contrast occurs naturally and is related to variations in thechemical environments around water protons (major contributor) and lipidprotons (usually minor). Chemical agents have been used to enhance thisnatural contrast. The one most widely tested clinically is theparamagnetic metal ion, gadolinium (Gd⁺³) (Rungs et al. (1983) Am. J.Radiol V 141, p 1209 and Weinman et al. (1984) Am. J. Radiol V 142, p619). Although gadolinium shortens both the T1 and T2 times, at thelower doses used for clinical imaging, the T1 effect generallypredominates and the image becomes brighter. Also, the rf pulse sequencecan be programmed to accentuate T1 changes and diminish those due to T2(Rungs et al. (1983) Am. J. Radiol V 141, p 1209). Hence, "T1-weighted"enhancement can be achieved by selecting the most favorable Gd dose andrf pulse sequence.

The shortening of proton relaxation times by Gd is mediated bydipole-dipole interactions between its unpaired electrons and adjacentwater protons. The effectiveness of Gd's magnetic dipole drops off veryrapidly as a function of its distance from these protons (as the sixthpower of the radius) (Brown (1985) Mag. Res. Imag. V 3, p 3).Consequently, the only protons which are relaxed efficiently are thoseable to enter Gd's first or second coordination spheres during theinterval between the rf pulse and signal detection. This ranges from 10⁵to 10⁶ protons/second ((Brown (1985) Mag. Res. Imag. V 3, p 3). Still,because Gd has the largest number of unpaired electrons (seven) in its4f orbital, it has the largest paramagnetic dipole (7.9 Bohr magnetons)and exhibits the greatest paramagnetic relaxivity of any element (Rungeet al. (1983) Am. J. Radiol V 141, p 1209 and Weinman et al. (1984) Am.J. Radiol V 142, p 619). Hence, Gd has the highest potential of anyelement for enhancing images. However, the free form of Gd is quitetoxic. This results in part, from precipitation at body pH (as thehydroxide). In order to increase solubility and decrease toxicity, Gdhas been chemically chelated by small organic molecules. To date, thechelator most satisfactory from the standpoints of general utility,activity, and toxicity is diethylenetriamine pentaacetic acid (DTPA)(Runge et al. (1983) Am. J. Radiol V 141, p 1209 and Weinman et al.(1984) Am. J. Radiol V 142, p 619). The first formulation of thischelate to undergo extensive clinical testing was developed by ScheringAG-Berlex Imaging according to a patent application filed in WestGermany by Gries, Rosenberg and Weinmann (DE-0S 3129906 A 1 (1981). Itconsists of Gd-DTPA which is pH-neutralized and stabilized with theorganic base, N-methyl-D-glucamine (meglumine). The Schering-Berlexagent is nearing completion of Phase III clinical testing at selectedcenters across the United States and abroad. The results of preliminarystudies indicate that almost all human brain tumors undergo significantenhancement (Felix et al. (1985) Proc. Soc. Mag. Res. Med. V 2, p 831and K. Maravilla, personal communication). These include metastaticcarcinomas, meningiomas, gliomas, adenomas and neuromas. Renal tumorsare also enhanced satisfactorily (Lanaido et al. (1985) Proc. Soc. Mag.Res. Med. V 2, p 877 and Brasch et al. (1983) Am. J. Radiol. V 141, p1019). The Schering-Berlex formulation is projected to be available forgeneral clinical use in 1987.

Despite its satisfactory relaxivity and toxicity, this formulation hasfour major disadvantages.

(1) Chelation of Gd markedly decreases its relaxivity (by 1/2 an orderof magnitude). This happens because chelators occupy almost all of Gd'sinner coordination sites which coincide with the strongest portion ofthe paramagnetic dipole (Koenig (1985) Proc. Soc. Mag. Res. Med. V 2, p833 and Geraldes et al. (1985) Proc. Soc. Mag. Res. Med. V 2, p 860).

(2) Gd-DTPA dimeglumine, like all small paramagnetic metal chelates,suffers a marked decrease in relaxivity at the higher radio frequenciesused clinically for proton imaging (typically 15 MHz) (Geraldes et al.(1985) Proc. Soc. Mag. Res. Med. V 2, p 860).

(3) Due to its low molecular weight, Gd-DTPA dimeglumine is cleared veryrapidly from the bloodstream (1/2 in 20 minutes) and also from tissuelesions (tumors) (Weinman et al. (1984) Am. J. Radiol V 142, p 619).This limits the imaging window (to ca. 30 to 45 minutes); limits thenumber of optimal images after each injection (to ca. 2); and increasesthe agent's required dose and relative toxicity.

(4) The biodistribution of Gd-DTPA is suboptimal for imaging of body(versus brain) tumors and infections. This is due to its small molecularsize. Intravenously administered Gd-DTPA exchanges rapidly into theextracellular water of normal tissues, as well as concentrates in tumorsand infections. This is facilitated by an absence in body organs, of the"blood-brain" vascular barrier which partly restricts the exchange ofGd-DTPA into the extracellular water of normal (versus diseased) brain.The result in body organs, is a reduced difference in the concentrationof Gd-DTPA between normal and diseased regions of tissue, and hence,reduced image contrast between the normal and diseased regions of theorgan. Also a disproportionate quantity (>90%) of Gd-DTPA is sequesteredvery rapidly in the kidneys (Weinman et al. (1984) Am. J. Radiol V 142,p 619). Of much greater interest to body MRI, are the abdominal sitesinvolved in the early detection and staging of tumors (particularly theliver, and also the spleen, bone marrow, colon and pancreas).

Three approaches have been taken in attempts to overcome thesedisadvantages.

(1) Alternative, small chelating molecules have been tested. These makeGd more accessible to water protons but still chelate the metal with asufficient affinity to potentially control its toxicity in vivo. Themost effective of these chelators is DOTA, the polyazamacrocyclicligand, 1,4,7,10-tetraazacyclododecane-N,N',N"-tetraacetic acid(Geraldes et al. (1985) Proc. Soc. Mag. Res. Med. V 2, p 860). Itsrelaxivity is approximately 2 times greater than that of Gd-DTPA over awide range of Larmor frequencies. However, it is still less active thanfree Gd.

(2) Gd and Gd-chelates have been chemically conjugated tomacromolecules, primarily the proteins, albumin (Bulman et al. (1981)Health Physics V 40, p 228 and Lauffer et al. (1985) Mag. Res. Imaging V3, p 11), asialofetuin (Bulman et al. (1981) Health Physics V 40, p228), and immunoglobulins (Lauffer et al. (1985) Mag. Res. Imaging V 3,p 11 and Brady et al. (1983) Soc. Mag. Res., 35 2nd Ann. Mtg., Works inProgress, San Francisco, Calif.). This increases the relaxivity of Gd byslowing its rate of molecular tumbling (rotational correlation time)(Lauffer et al. (1985) Mag. Res. Imaging V 3, p 11). This improvescoupling of the energy-transfer process between protons and Gd (Geraldeset al. (1985) Proc. Soc. Mag. Res. Med. V 2, p 860, Lauffer et al.(1985) Mag. Res. Imaging V 3, p 11 and Brown et al. (1977) BiochemistryV 16, p 3883). Relaxivities are increased by multiples of 5 to 10relative to Gd-DTPA (when compared as R1=1/T1 values at 1 millimolarconcentrations of Gd) and by multiples of 2.5 to 5.0 (when compared asthe molarities of Gd required to produce a specified decrease in the T1relative to a control solution (physiologic saline).

The reasons for using the latter method of comparison are that 1)millimolar concentrations of Gd are never achieved in vivo--actualtissue concentrations achieved in the usual image enhancement arebetween 20 and 100 micromolar Gd; 2) the slopes of R1 graphs arefrequently nonparallel for different enhancing agents; 3) the secondmethod allows agents to De compared according to the more customarymeans of chemical activity ratio, in other words, as the concentrationrequired to produce a specified percentage decrease in the T1 (or T2)relaxation time. Although R1 data are supplied below for the purpose ofliterature comparisons the second method is considered preferable and isthe one used for internal comparisons of potency throughout theremainder of the application. The large drawback of conjugating DTPA toprotein carriers for use in NMR image enhancement is that it has beendifficult to stably conjugate more than 5 DTPA's (and hence Gd's) toeach albumin molecule (Bulman et al. (1981) Health Physics V 40, p 228,Lauffer et al. (1985) Mag. Res. Imaging V 3, p 11 and Hnatowich et al.(1982) Int. J. Appl. Radiat. Isot. V 33, p 327 (1982).

Comparably low substitution ratios (normalized for molecular weight)have been reported for immunoglobulins (Lauffer et al. (1985) Mag. Res.Imaging V 3, p 11 and Brady et al. (1983) Soc. Mag. Res., 2nd Ann. Mtg.,Works in Progress, San Francisco, CA) and fibrinogen (Layne et al.(1982) J. Nucl. Med. V 23, p 627). This results from the relativedifficulty of forming amide bonds, the comparatively low number ofexposed amino groups on typical proteins which are available forcoupling, and the relatively rapid hydrolysis of DTPA anhydride couplingsubstrate which occurs in the aqueous solvents required to minimizeprotein denaturation during conjugation (Hnatowich et al. (1982) Int. J.Appl. Radiat. Isot. V 33, p 327 (1982) and Krejcarek et al. (1977)Biochem. Biophys. Res. Comm. V 77, p 581). The overall effect of thesesuboptimal conditions is that a very large dose of carrier material isrequired to achieve significant in vivo effects on MR images. At thishigh dose, the carrier produces an unacceptable acute expansion of therecipient's blood volume by an osmotic mechanism. Indeed, lowsubstitution ratios have generally limited the use of suchprotein-chelator-metal complexes to the more sensitive (low-dose),radiopharmaceutical applications (Layne et al. (1982) J. Nucl. Med. V23, p 627).

An attempt to overcome this low substitution ratio has been made byconjugating DTPA to the non-protein carrier, cellulose (Bulman et al.(1981) Health Physics V 40, p 228), however the chemical method employedresults in continued suboptimal substitution of DTPA to carrier, thenonbiodegradability of cellulose and its water-soluble derivatives andthe reported molecular aggregation which results from organic-solventconjugation (in dimethylformamide) of CNBr-activated cellulose to thediaminohexyl spacer groups which link the carrier to DTPA, have renderedthis class of carrier-conjugates unacceptable for intravenousadministration at the doses required for MR image enhancement.

A very important consideration in the image enhancement of solid tumorsand inflammatory lesions by polymeric contrast agents is that, in orderfor these agents to extravasate (exit) efficiently from themicrocirculation into adjacent diseased tissues, they must be completelysoluble--e.g., not be contaminated by inter molecular or supramolecularmicroaggregates. Optimal tumor access and localization requires that themolecular size of such agents generally be less than approximately2,000,000 daltons (ca. 2 to 3 nanometers in molecular diameter), andpreferably less than 500,000 daltons (ca. 0.5 to 1 nanometer inmolecular diameter) (Jain (1985) Biotechnology Progress V 1, p 81). Forthis reason, with rare exceptions (see Example 6, below) the particulateand microaggregate classes of contrast agents (which comprise theliposomes, colloids, emulsions, particles, microspheres andmicroaggregates, as described below) do not concentrate efficiently inmost solid tumors and inflammatory lesions. Instead, followingintravenous administration, these supramolecular-sized agents: a) arefirst circulated in the bloodstream for relatively short intervals (25minutes to 24 hours, depending on size), potentially allowing directimage enhancement of the blood pool (plasma compartment); and b) aresubsequently cleared by specialized (phagocytic) cells of thereticuloendothelial tissues (liver, spleen and bone marrow), potentiallyallowing selective enhancement of these normal tissues, but producingindirect (negative) enhancement of lesions within these tissues (due toexclusion of the agents from the diseased regions). Additionally,following installation into the gastrointestinal tract and other bodycavities, these particulate and microaggregate classes of agents canproduce direct image enhancement of the fluids within these cavities,and thereby potentially delineate mass lesions which encroach upon thelumens and cavities. Both microspheres and microaggregates aresupramolecular in size. The microaggregate class of agents is produced(intentionally or unintentionally) by either a) molecular cross-linkingof individual polymer molecules or b) secondary aggregation ofpreviously singlet (soluble) polymers, as induced by charge attractionor hydrophobic bonding mechanisms. It is distinguished from themicrosphere class of agents by virtue of its smaller particle size,which ranges from approximately 2,000,000 daltons (ca. 2 to 3 nanometersin diameter) to 0.1 micrometers (=100 nanometers in diameter). It isimportant to note that microaggregates are cleared byreticuloendothelial phagocytes with significantly less efficiency andrapidity than are microspheres. In general, this property makesmicroaggregates a less preferred class of agents for visualizing theliver, spleen and bone marrow under the usual conditions of clinicalimaging, for which prompt post-injection contrast enhancement isrequired.

(3) Gd-DTPA has been entrapped in liposomes (Buonocore et al. (1985)Proc. Soc. Mag. Res. Med. V 2, p 838) in order to selectively enhanceimages of the reticuloendothelial organs (liver, spleen and bone marrow)and potentially the lungs. Liver clearance is mediated by phagocytic(Kupffer) cells which spontaneously remove these small (0.05 to 0.1 um)particles from the bloodstream (Buonocore et al. (1985) Proc. Soc. Mag.Res. Med. V 2, p 838). (Particles larger than 3 to 5 um are selectivelylocalized in the lungs due to embolic entrapment in lung capillaries.) Arecent report indicates that the small-sized Gd-liposomes produceeffective decreases in liver T1's ( as determined spectroscopicallywithout imaging) (Buonocore et al. (1985) Proc. Soc. Mag. Res. Med. V 2,p 838). Also, insoluble Gd-DTPA colloids have recently been reported toenhance MR images of rabbit livers under in vivo conditions (Wolf et al.(1984) Radiographics V 4, p 66). However, three major problems appear tolimit the diagnostic utility of these devices. The multilamellar, lipidenvelopes of liposomes appear to impede the free diffusion of waterprotons into the central, hydrophobic cores of these carriers, asassessed by the higher doses of Gd required for in vitro relaxivitiesequivalent to Gd-DTPA dimeglumine (Buonocore et al. (1985) Proc. Soc.Mag. Res. Med. V 2, p 838). This increases the relative toxicity of eachGd atom.

Even more importantly, these same lipid components cause the carriers tointeract with cell membranes of the target organs in a way which leadsto a marked prolongation of tissue retention (with clearance times of upto several months) (Graybill et al. (1982) J. Infect. Dis. V 145, p. 748and Taylor et al. (1982) Am. Rev. Resp. Dis. V 125, p 610); and G.Kabala, personal communication). Two adverse consequences result. First,image enhancement does not return to baseline in a timely fashion. Thisprecludes re-imaging at the short intervals (ca. I to 3-weeks) needed toassess acute disease progression and treatment effects. Second,significant quantities of the liposomally entrapped Gd-DTPA may betransferred directly into the membranes of host cells (Blank et al.(1980) Health Physics V 39, p 913; Chan et al. (1985) Proc. Soc. Mag.Res. Med. V 2, p 846). This can markedly increase the cellular retentionand toxicity of such liposomal agents. The consequences for Gd toxicityhave not yet been reported. Protein (albumin) microspheres withentrapped Gd and Gd chelates have been prepared and determined by thepresent applicant and others (Saini et al. (1985) Proc. Soc. Mag. Res.Med. V 2, p 896) to have only modest effects on T1 relaxivity in vitro.This is because most of the Gd as well as other entrapment materials(Widder et al. (1980) Cancer Res. V 40, p 3512) are initiallysequestered in the interior of these spheres and are released veryslowly as the spheres become hydrated (with t1/2's of hours) (Widder etal. (1980) Cancer Res. V 40, p 3512). This phenomenon has been found bythe present applicant to markedly reduce the acute (30-to-90-minute)relaxivity of each Gd atom to approximately 1/10th that of Gd-DTPAdimeglumine. Hence, both the quantity of carrier material and thetoxicity of Gd are both unnecessarily high.

Emulsions of insoluble, gadolinium oxide particles have been injectedinto experimental animals with significant image-enhancing effects onthe liver (Burnett et al. (1985) Magnetic Res. Imaging V 3, p 65).However, these particles are considerably more toxic than any of thepreceding materials and are inappropriate for human use. Because of thesignificant disadvantages of existing MR image contrast agents, thepresent applicant has formulated improved, second-generation prototypeagents with reduced toxicity, increased selectivity of tumor and organuptake, as well as a significant potential for enhancing blood flowimages.

Many of the advantages shown for the present developments concerning NMRimage-enhancing agents (also referred to herein as NMR contrast agentsor MR (magnetic resonance) contrast agents) are also expandable to otherareas. For example, high-field NMR surface-coil spectroscopy of ¹ H¹³ C,¹⁹ F, ²³ Na, and ³¹ p nuclei in spacially localized tissue volumes isgaining in importance and is starting to be applied experimentally tothe noninvasive clinical monitoring of genetic and metabolic disorders;myocardial infarcts and metabolism; brain, liver and tumor metabolism;drug distribution and metabolism; blood flow and tissue perfusionmeasurements; and temperature monitoring in regional hyperthermia.Gadolinium and related agents can produce characteristic changes in theNMR spectrum of adjacent NMR-susceptible nuclei. These changes include:modulation of emission peak positions, widths, intensities, andrelaxation rates (which affect intensity). Hence, perturbation ofspectra by such chemical shift-relaxation agents can be used to localizeand identify the source of NMR signals with respect to organ location,tissue compartment (intravascular versus extravascular), cell typewithin the tissue, and potentially, the specific metabolic pathwayswithin cells which are altered by drugs and disease. Also in certainsituations, ultrasound imaging or body scanning of radioisotopicemissions is particularly useful in achieving insight into internalstructures. The radioisotopic emissions most frequently scanned arethose of metallic radioisotopes emitting gamma particles, however,positron emission tomography is expaexperiencing increased clinicsl use.The molecular formulation and mode of administering these radioisotopicmetals will have significant consequences on the internal localizationand body half-life of these radioisotopes, potentially leading toincreased diagnostic usage of these ultrasound images and emissionscannings.

The present invention includes an image-enhancing or spectral-shiftagent comprising a biodegradable, water-soluble polymer, synthetic orderived from natural sources and having repeating hydrophilic monomericunits with a high frequency of amino or hydroxyl groups. This agent alsoincludes chelating agents comprising functional groups bound to anamino, quaternary ammonium or other reactive nitrogen group; hydroxyl;carboxy; sulfhydryl; sulfate or sulfonium group of the monomeric units.These chelating agents have a formation constant for divalent ortrivalent metal cations of at least about 10⁸ (and typically >10¹³) atphysiological temperature and pH. The conjugation of chelating groups tothe polymer (or to form the copolymer) is carried out under chemicalconditions and in a solvent which yields a completely soluble (singlet)form of the carrier and avoids significant contamination bymicroaggregates. The molar ratio of chelating agent/monomeric unit ispreferably between about 1/5 and about 1/25. The molar ratio ofchelating agent/monomeric unit is preferably between about 1/5 and about1/25. This image-enhancing agent is biodegradable to intermediarymetabolites, rapidly excretable chelates, polymers, oligomers, monomersor combinations thereof, all of which have low toxicity and are clearedoverwhelming by the renal route. The term "low toxicity" used hereinmeans having little significant toxic effects at usable dosages of theimage-enhancing agents.

These image-enhancing agents may further comprise a paramagnetic metal,transition element or rare-earth ion for enhancement of the images orspectra arising from induced magnetic resonance signals. As definedherein, the term "metal ions" refers to any of these materials as beingcapable of forming positively charged ions. The polymeric (ormicrosphere--see below) nature of these agents, is designed to produce asubstantial increase in the NMR potency of each paramagnetic metal ion,compared to small metal chelates.

Images resulting from scanning of gamma or positron particle emissionsmay be enhanced when the image-enhancing agent of the present inventioncomprises a radioisotopic metal, transition element or rare-earth ion(or oxides of the preceding entities) emitting gamma positron particles.

Images resulting from ultrasound scanning may be enhanced by modifyingthe native tissue reflectivity (velocity) of high frequency sound waves,when the image-enhancing agent of the present invention comprises one ofthe relatively dense, nonradioactive metals or metal ions.

The physical conversion of these soluble image-enhancing agents intomicrospheres (greater than or equal 100 nanometers in diameter). allowsfurther internal targeting of the image-enhancing agents, either by oraladministration to the gastrointestinal tract, or by intravenousadministration to organs with phagocytic capabilities (principallyliver, spleen and bone marrow).

The alternative conversion of these soluble image-enhancing agents intomicroaggregates (3 to 100 nanometers in diameter) also allows theirinternal targeting to phagocytic organs, but with substantially lessefficiency and rapidity than occurs with microspheres.

Images of the internal structures of an animal may be obtained by a widevariety of means known to those skilled in the art. In one general fieldof imaging, the administration of metal, transition-element andrare-earth containing markers is utilized. These markers, because of thephysical properties of their metal components, may be used to enhancethe quality of images produced by numerous means.

Among the image-producing means where metallic transition element andrare-earth markers may be advantageously used are magnetic resonance(MR) imaging, ultrasound imaging and scanning of gamma and positronparticle emissions. Foremost as a preferred embodiment of the presentinvention is the enhancement of images produced by nuclear magneticresonance (NMR) imaging of whole animals or portions thereof. The termsmagnetic resonance (MR) imaging and nuclear magnetic resonance (NMR)imaging are used herein as equivalent terms.

The present invention comprises novel ways to entrap metal- orparamagnetic metal-chelate complexes in biodegradable, hydrophilicpolymeric microcarriers. First, the chelate is chemically conjugated inlarge numbers to hydrophilic polymers such as long-chain dextrans (1-6linked, soluble, moderately branched polymers of glucose). Thesehydrophilic polymers are biodegradable and water-soluble. They areeither synthetic or derived from eukaryotes, procaryotes, hybridorganisms, or plants and comprise repeating hydrophilic monomeric unitshaving amino, hydroxyl or sulfate groups. They may be furtherdeveratized to contain carboxylic acid, sulfonium or sulfhydryl groups;or quaternary ammonium or other reactive nitrogen groups. The negativelycharged, naturally occurring, repeating hydroxyl or sulfate groups maycontribute to the stabilization of binding of positively charged Gdions, over and above the stability of binding conferred by thecovalently conjugated chelators.

The image-enhancing agents of the present invention comprise chelated orchelating agents having functional groups bound to hydroxyl, amino,quaternary ammonium (or other nitrogen functional group) , carboxy,sulfhydryl, sulfonium or sulfate group of the monomeric units of thepolymer. These chelating agents are further defined as having formation(stability) constants for divalent or trivalent metal cations of atleast about 10⁸ and typically greater than 10¹³ at mammalianphysiological pH and temperatures.

The whole image-enhancing agents described above are characterized asbeing biodegradable by mammals to intermediary metabolites or excretablechelates, polymers, oligomers, monomers or combinations thereof, all ofwhich have low toxicity.

Chelating agents having the properties described above have then the twobasic properties of affinity for divalent or trivalent metals and alsothe ability to bond to one or more reactive groups as listed 2paragraphs above, which themselves are bound to the polymer.Particularly preferred chelating agents of the present invention includeEDTA (ethylenediaminetetraacetic acid); DTPA(diethylenetriaminepentaacetic acid); TTHA(triethylenetetraaminehexa-acetic acid); and DOTA(1,4,7,10-tetrazacyclododecane-N,N',",N'"-tetraacetic acid).

A particularly preferred image-enhancing agent of the present inventioncomprises dextran polymer and DTPA chelating agent wherein the method ofconjugation results in a polymer-chelate which is completely watersoluble--e.g. avoids microaggregation--(see below). This particularlypreferred agent, when in combination with gadolinium has been found tovery effectively enhance internal (in vivo) images arising from inducedmagnetic resonance signals. Alternative elements (ions) for use in MRIcould include those of atomic numbers 21 through 29 and 57 through 70,with particular emphasis on numbers 24-29 and 62-69.

The polymer of the image-enhancing agents described herein is preferablya polysaccharide or oligosaccharide and most preferably dextran.Polyamino substances, poly-L-lysine, for example, are usable but notgenerally preferred because of their net polymeric (closely spaced)positive charges at a physiological pH, although in conceivablecircumstances this type of polymer could be desirable. In all cases, thepolymers of the present invention should be biodegradable. This term"biodegradability", as used herein, indicates that internally availablemammalian enzymes or conditions lead to the breakdown of the polymer,particularly to an excretable and non-toxic form. Thus non-biodegradablepolysaccharides such as cellulose and its water-soluble derivatives arenot preferred for the practice of the present invention.Biodegradability may indicate further that mammalian enzymes orconditions lead to cleavage of the chemical bond which attaches themetal chelator to the polymer or to the alternating monomeric units orthe copolymer.

In size, the polymers of the present invention should have molecularweights of between 1,000 and 2,000,000 daltons. A more preferable sizerange for most uses is between about 40,000 daltons and about 75,000daltons, this range representing a frequent optimum for the hybridobjectives of, amplifying the relaxivity of each Gd, allowingextravasation of an initially intravascular agent, and localization ofthis agent in tumors and inflammatory lesions, and ofslightly-to-moderately delaying or otherwise modifying the renalexcretion of these polymeric agents relative to lower molecular weightagents such as Gd:DTPA (dimeglumine).

The functional groups of the chelating agents are preferably bound tothe monomeric units of the polymer by a covalent linkage, although incertain cases a strong noncovalent bond may be usable. The mostpreferable covalent bond of chelating agent to polymer is an esterlinkage, due to its ease of formation, adequate stability for biologicaltargeting, and optimal susceptibility to enzymatic cleavage forsubsequent (post-imaging), clearance of the metal chelates from targetcells and from the body.

The method used to conjugate the chelator to the carrier is of criticalimportance from the standpoint of agent solubility, and hence,biodistribution, performance, and acceptability of these solublepolymers in vivo. In order to obtain high substitution ratios of thechelator to carrier, and most importantly to avoid cross-linking thepolymer singlets (prevent microaggregation), as well as to simplifychelator conjugation, minimize reagent expense, and facilitatesubsequent biodegradation in vivo, conjugation of chelator groups whenperformed in the usual, side-chain configuration, is preferably carriedout according to a one-step method which requires aqueous-phase couplingof the chelator's di-anhydride substrate, at a physiologic pH of lessthan 8.5, to carrier dextran of the desired molecular size. A carriersize of greater than approximately 10,000 to 20,000 daltons is preferredin order to provide the additional advantage of limiting the agentsinitial biodistribution almost exclusively to the blood vascularcompartment (Grotte (1956) Acta Chirurgica Scandanavia V 211(supplement), p 5). These features are required to: a) minimize theagent's access to the body's normal extracellular fluid, therebyproviding maximal selectivity towards tumor imaging; b) allow maximalextravasation of the agent into tumors and inflammatory lesions (due tothe capacity of completely soluble--e.g., nonaggregated--polymers ofthis size range to emigrate through the moderately enlarged "pores"which are present in tumor versus normal microvessels; and c) minimizeclearance by reticuloendothelial phagocytes (which would reduce agentpotency by removing it from the blood compartment, making it unavailableto tumors. These methodologic considerations are of utmost importancefor maximizing agent usefulness, acceptability, and potency in vivo;optimizing renal clearance; and minimizing body toxicity.

Physical conversion of the soluble polymeric agent to microaggregates ispossible but not preferred. However, it does occur inadvertently, and itcan be achieved intentionally by either of the following chemicalmethods; a) by carrying out the chelator-carrier conjugation in anynonprotonating (organic) solvent. This facilitates the cross-linking(microaggregation) of adjacent polymer molecules because waterhydrolysis of the chelator's second (unreacted) anhydride group cannotoccur as rapidly as required after the first anhydride has reacted witha dextran molecules or b) by exposing the soluble (nonaggregated) formof the polymer-chelator-metal complex to pH's of approximately 8.5 orgreater, which can produce microaggregation due to electrostatic(charge) effects.

A second preferable physical form of the image-enhancing agents of thepresent invention is one of microspheres. The preferable size range ofthese microspheres is between about 0.1 um and about 250 um. An NMRimage-enhancing agent may be formed into microspheres, either before orafter the addition of a paramagnetic metal ion such as that ofgadolinium. The resultant microspheres, when administered at diametersless than 3 um to a mammal by intravenous injection have been found tobe taken up by organs such as the liver, spleen and bone marrow. Thusthe normal tissue components of these organs, for example, are renderedselectively preferentially able to yield improved images arising frominduced magnetic resonance signals. Because of differential organclearance, microspheres of sizes 0.1 to 3.0 um are preferable for imageenhancement of liver, spleen and bone marrow; and microspheres of sizes3 to 250 um are preferable for image enhancement of lung.

Another significant aspect of the present invention may involve thefurther rapid coupling of chelate-polymer image-enhancing agentsthemselves to proteins such as hormones, polyclonal or monoclonalantibodies or to a substance which secondarily binds either native orderivatized antibodies, (e.g., protein A, biotin or avidin). Thiscoupling may involve, for example, sodium periodate oxidation of vicinalsugar hydroxyl Groups such as those of a polysaccharide and reduction ofSchiff-bases by sodium borohydride to related, stable, covalent bondswith protein amino groups. The specific binding characteristics ofantibodies, when combined with multiply chelatively bound metal ions maybe used to produce specific localization of large numbers ofparamagnetic or particle-emitting ions within internal targets ofinterest, thus amplifying greatly the signal-modulating effects of eachspecifically localized substance and also preserving or improving theantibody-binding specificity, affinity and avidity.

The image-enhancing agents of the present invention are also usable toenhance images being produced from the scanning of gamma and positronparticle emissions and by ultrasound detectors. In this usage, most ofthe general principles of NMR image-enhancement--except agentdose--apply, the major difference being that now the chelated metal ionis respectively, a radioisotope which emits gamma particles, or one ofthe relatively nonradioisotopes which alters the velocity of transmittedand reflected ultrasound waves. Preferable radioisotopic metals include⁵¹ chromium, ⁶⁸ gallium, ¹¹¹ indium, ⁹⁹ technetium and its oxides.Useful ultrasound metals (ions) include those of atomic number 20(calcium), 25 and 26 (manganese and iron, respectively), preferably57-70 (the rare earth series), and optimally 64 (gadolinium).

A general object of the present invention comprises formulation and useof an image-enhancing agent, most particularly for images induced bymagnetic resonance. This image-enhancing agent comprises a chelatingagent bound to a water-soluble biodegradable polymer. The agent may beutilized in soluble form or as microspheres. In soluble form theimage-enhancing agent, when administered to an animal, is primarilydistributed in circulating blood, kidney and especially at sizes ofabout 20,000 to 500,000 MW, also has the capacity to exit the vascularcompartment selectively in regions of tumors and inflammations and focusthese tissue lesions.

In small microsphere form, the image-enhancing agent, uponadministration by injection into animals, is preferentially cleared byand redistributed to liver, spleen and bone marrow. Upon oraladministration, microspheres may be introduced into the gastrointestinaltract for image visualization thereof. Upon subsequent biodegradation inthe small and large intestine, the preferred metal (e.g., gadolinium)will form insoluble oxides which are not absorbed internally, and aretherefore nontoxic.

The acute enhancement of blood flow images, for example in the heart orcerebral vessels, may be accomplished with the soluble polymericimage-enhancing agent and is even more efficiently performed with themicrosphere form.

A significant advantage of image enhancement with polymeric andmicrosphere chelators, in connection with the marginally toxic metals,particularly paramagnetic ones such as gadolinium, is a furtherreduction of necessary metal dose and decrease in toxicity over thatwhich can be achieved by simple (low molecular weight) chelating agentsalone.

The relatively rapid biodegradation and metal clearance times, and theresultant shorter re-imaging intervals are particular advantagesinvolved with the present invention relative to other polymeric andparticulate metal chelates and complexes.

The image-enhancing agents of the present invention, in soluble ormicrosphere form, are readily reconstituted for animal and patientadministration. This reconstitution involves a simple vortex-typemixing, as compared to sonification in detergents used for protein-basedmicrospheres.

The image-enhancing agents of the present invention are easily usable inany detection or imaging system involving administration of divalent ortrivalent metallic marker ions. The appropriate metal need only be addedto the polymer-chelate complex at pH's consistent with stable chelationbinding (typically ≧3.0 to 3.5) or the polymer-chelate complex beformulated as heat-stabilized or variously coated microspheres toprotect against dechelation during passage through the more acidicenvironment of the stomach (typically pH=1.0-2.0).

The image or spectral enhancing agents of the present invention allowshorter image acquisition times for satisfactory internal resolutions.Shorter image acquisition times are generally adequate to producesatisfactory internal images because of the greater signal enhancementand image contrast produced per unit of chelated marker and total agent.

The potential for specific location of large numbers of marker metalions by small numbers of monoclonal antibodies, nonpeptide and peptidehormones and receptor-binding substances tagged with one or moreimage-enhancing agents is contemplated as a major diagnostic advantageand future use.

Additionally, because of the high contrast, moderate prolongation oflesional residence times (soluble polymers) and liver, spleen and bonemarrow residence times (microspheres) (of several hours versus minutesfor small molecular forms) the use of the present image-enhancing agentsallows an increased number of serial images to be obtained in theenhanced mode after a single administration of agent.

From a chemical point of view, some advantages of the present inventionmay be summarized as follows. When NMR image-enhancing agents comprisepare magnetic metals such as gadolinium ion, each gadolinium ionexhibits an increased relaxivity for adjacent magnetic nuclei (e.g.protons) and hence gives greater T1 signal enhancement. This increasedrelaxivity is related to an increased dipolar correlation time of Gd dueto slower molecular rotation of polymeric Gd, the hydrophilic polymer(which becomes completely hydrated and allows rapid on-off binding(hence relaxation) of adjacent paramagnetic nuclei (protons)). Spacergroups are not required between the metal chelates and the polymericcarrier in order to obtain optimal paramagnetic relaxation potencies,however, they could be introduced if deemed advantageous for otherpurposes. When microspheres are used, the small microsphere size allowsaccess of hydrated magnetic nuclei to virtually all of the chelatedparamagnetic ions.

The chemically defined nature of preferred chelator-polymer combinationsallows ready batch-to-batch uniformity for improved pharmaceuticalformulations and a likely greater ease of FDA approval.

Many preferred components of the present invention, such as certaindextrans (40,000 and 70,000 MW forms), DTPA and Gd (as the DTPAchelate), for example have already separately achieved preliminary orfinal FDA approval.

For parenteral administration, these agents are preferably formulated asa sterile, physiologically balanced, aqueous solution (or suspension),whose pH for purposes of intravenous administration is either a)approximately 6.0 to 7.5 for biodistribution and localization of thesoluble polymer, microspheres, or preformulated microaggregates; or b)8.5 or greater for biodistribution and localization of microaggregateswhich were formed after conjugation of the chelator to soluble polymer,by electrostatic aggregation. Alternatively, these agents may belyophilized and supplied in the dried from for reconstitution inphysiologic solutions just prior to administration. For gastrointestinaladministration (oral or rectal), or injection into body cavities (suchas the bladder, uterus, Fallopian tubes, nasal sinuses orventriculo-cerebrospinal system), these agents may be formulated as aphysiological solution (or suspension) which contains additionalsubstances to increase the viscosity or osmolality. For oraladministration, the agents may be further formulated according tostandard pharmaceutical methods, as uncoated or coated, micro- ormacrotablets, in order to provide additional protection against theacidic pH of the stomach, and thereby avoid the release of chelatedmetal ions, which typically occurs at gastric pH's. Other additives,such as flavorings and colorings may be also incorporated according tostandard pharmaceutical procedures.

For parenteral administration, the concentration of total active agent(polymer-metal chelate) will be between 0.1% and 30% (weight/volume),typically between 5% and 25%, and preferably 20%. Doses of the solublepolymeric and microsphere agents will vary depending on the paramagneticmetal and the route of administration. The following doses are given forintravenous administration For tumor image enhancement with thepreferred embodiment, soluble Gd-DTPA-dextran 70, the dose will bebetween 0.01 and 0.075 millimoles of Gd per kilogram body weight, withoptimal image enhancement occurring typically at or below 0.03millimoles of Gd per kilogram. For liver, spleen and/or bone marrowenhancement with the preferred embodiment, microsphere Gd-DTPA-dextran70, the dose will be between 0.008 and 0.05 millimoles of Gd perkilogram, with optimal image enhancement occurring typically at or below0.01 millimoles per kilogram. For enhancement of the cardiovascularblood pool, the optimal dose of soluble Gd-DTPA-dextran 70 andmicrosphere Gd-DTPA-dextran 70 will occur, respectively, at or below0.08 and 0.04 millimoles Gd per kilogram.

The following examples are presented to illustrate preferred embodimentsof the present invention and their use in MR imaging. These examples arenot intended to limit the scope of the present invention in any wayunless otherwise so stated in the claims later appended hereto.

EXAMPLE 1 COMPARISON OF SPECIFIC ACTIVITIES AND R1 RELAXIVITIES OF IRONCOMPLEXED TO DEXTRAN 70 AND LOW MOLECULAR WEIGHT IONIC IRON COMPOUNDS(FERRIC NITRATE AND FERRITIN-IRON)

Fe⁺³ as the dextran-iron oxide (in which the iron is complexed looselyto the hydroxyl groups of dextran), was obtained as "Proferdex" (20%iron, w/w) (Fisons Pharmaceuticals) and tested, both before and afterextensive dialysis (to remove loosely bound iron), for NMR T1-enhancingactivity in vitro (using an IBM PC20 Minispectrometer, 20 MHz). Thisresult was compared with those of Fe⁺³ in the forms of ferric nitrate(Fe(NO₃)₃.9H₂ O, obtained from Sigma Chemicals, St. Louis, Mo.) andiron-ferritin (obtained as apoferritin saturated with iron at 8.5% (w/w)(Polysciences, Inc.) in which Fe⁺³ is entrapped inside the core cavityof the apoprotein at up to a 4,500:1 molar ratio to the protein, but inwhich the rotational correlation time of the ionic iron is not prolongedby virtue of direct conjugation to the protein. Following Table 1describes many of the above results.

                  TABLE 1                                                         ______________________________________                                                  Fe.sup.+3 concentration                                                                        R1                                                           (ug/ml) producing a 50%                                                                        relaxivity)                                        Compound  decrease in water T1                                                                           (1/mmol × sec)                               ______________________________________                                        Dextran-Fe                                                                              19               0.817                                              Ferric nitrate                                                                          42               1.476                                              Fe-ferritin                                                                             100              0.541                                              ______________________________________                                    

As assessed by the more accurate method (of Fe⁺³ concentration producinga 50% decrease in the water proton T1 time), the loose conjugation ofiron to macromolecular dextrans increased the specific paramagneticactivity of Fe+3 by a multiple of 2.21 relative to ferric nitrate (42/19ug/ml)--lower Fe concentrations indicate a higher ¹ H relaxivity per Featom. This increased T1-enhancing potency dextran-iron results from theslower rotational correlation time of iron following complexation to itslarger, dextran carrier. Ferritin-Fe was less potent than ferric nitrate(50% activity multiple=0.42, corresponding to 42/100 ug/ml). Thisreduced potency results from partial sequestration of ionic iron in thecore cavity of the apoferritin protein, which decreases the on-off rateof exchange of external water protons during the T1 relaxation interval.This potency decrease is observed even under conditions of a veryminimal diffusional barrier imposed by the monomolecular coat of theentrapment protein. It corroborates the even larger decreases ofrelaxation potency which were reported and observed above, forparamagnetic metals entrapped within liposomes and stabilized albuminmicrosperes.

Whereas the significant enhancement of potency observed for dextran iron("Proferdex") together with its macromolecular nature, suggested that itmight be a suitable prototype T1-contrast agent for intravenousadministration and restricted biodistribution (initially within theblood vascular compartment), extensive clinical trials (for treatment ofiron deficienty anemias) had indicated that its intravenous (versusintramuscular) administration was followed frequently by systemichypotension (Physicians Desk Reference (1986) p 1228). This was trueespecially if the agent was administered over an interval of severalminutes, as would be required for MR image enhancement. This in vivotoxicity was thought to result from rapid release of the weaklycomplexed-iron (oxide). To elucidate this, dextran-iron was extensivelydialyzed and the NMR T1 activity ratios of the entire molecules werecompared by the 50% concentration method (above). By this method, onlyapproximately 17.7% of the pre-dialysis iron remained complexed to thedextran carrier. This both explains the in vivo toxicity and indicateswhy dextran-iron (and by inference, other dextran-metal) oxide complexesare unlikely to represent preferred embodiments of the presentinvention. From these data, it was apparent that the preferredembodiment(s) for intravenous use would be more likely to comprisedextran carriers with covalently conjugated chelating groups which hadstability constants for metal chelation which were significantly higherthan those of dextran-iron oxide complexation (see following examples).However, dextran-iron could be of significant use for gastrointestinaland other parenteral applications in which iron release from the carrierwas less critical.

EXAMPLE 2 PREPARATION OF DTPA-DEXTRAN A. Conjugation In Aqueous Solvent

1. Preparation and Maintenance of Completely Soluble (Nonaggregated)Polymer Conjugates

The cyclic dianhydride of DTPA, prepared by the method of Eckelman etal. (J. Pharm. Sci. V 64, pp 704-706 (1975)), was obtained in a highlypure form from Calbiochem-Behring Corp. 6.0 g of the cyclic dianhydridewas added stepwise to 1.72 g of Dextran T70 (average MW 70,000 daltons,Pharmacia Chemicals) in a reaction solvent comprising HEPES buffer 115mg/100 cc distilled water, pH 7.0 to 8.0 (maximally). The reaction wascarried out with vigorous stirring at ambient temperatures for a 1 hrperiod with readjustment to pH 7.0 to 8.0 using NaOH, after eachsegmental addition of DTPA dianhydride. The dextran-DTPA product wasseparated from unconjugated DTPA by dialysis against 200 volumes ofdistilled water at pH 5.5. As assessed by molecular filtration, 97.8% ofthe dextran-DTPA product had a molecular weight of less than 100,000daltons and only 1.6% had a molecular weight greater than 300,000daltons. The dilute solution of dialyzed dextran-DTPA was concentratedto between 5% and 20% (w/v) by one of three methods: a) forced,filtered-air evaporation at room temperature (preferred); D; retentionover a nitrogen pressurized, 10,000 MW cutoff filter (AmiconCorporation); or c) lyophilization and reconstitution in physiologicsolutions. None of these methods produced a significant increase in theaverage molecular weight, as assessed by molecular filtration (above).This indicated that no significant inter molecular aggregation wasinduced by any of the three methods of concentration. In the first twomethods (a and b, above), concentrated salts and buffers were added asneeded, to render the final preparations physiologically acceptable forsubsequent injection. In all cases, care was taken to maintain the pH ator below 8.0 (generally between 6.5 and 7.0), in order to preventpost-conjugation aggregation of the dextran-DTPA. (See below forintentional preparation of microaggregates from this soluble polymer.)

Gadolinium in the form of GdCl₃. 7.05H₂ O (Alfa Laboratories, 2.1 g in10 cc distilled water was added to 1.38 g of the concentrateddextran-DTPA conjugate in distilled water adjusted to pH 5.5 with NaOH.Unbound gadolinium was removed from the dextran-DTPA gadolinium complexby molecular filtration through a 10,000 MW cutoff filter. Freegadolinium was monitored by standard complexometric titrations usingxylenol orange (Lyle et al. (1963) Talanta V 10, p 1177) and minimizedfor each preparation. Alternatively and preferred, the binding capacityof polymer was determined in advance and the quantity of Gd adjusted tobe exactly stoichiometric, leaving neither free Gd nor free polymericDTPA. This standard complexometric titration was also used to quantifytotal gadolinium of each preparation after oxidative acid hydrolysis ofthe organic matrix followed by neutralization of the released Gd. Asrecently prepared, one of every 12.2 sugar residues is conjugated to anactive DTPA ligand, for a total of 32 Gd-binding ligands per 389 Glucoseunits. (It is anticipated that significantly higher derivatizationratios can be achieved without producing significant carriercross-linking, by increasing the quantity of DTPA anhydride andmaintaining more continuous control over the reaction pH with a pHstat.) The in vitro T1 relaxivity, R1, in physiologic saline is greaterthan 100/(mM×sec) (IBM PC20 Minispectrometer, IBM Instruments). (Bycomparison, the R1 of GdC₁₃ was 3.03/(mM×sec). The osmolality of solubleGd-DTPA-dextran T70 is at or below 3590 mOsm/kg product, as determinedby the vapor pressure method (Wescor Model 5100B osmometer, WescorInstruments). As a final quality-control check, the concentratedproduct, Gd-DTPA-dextran, was tested in an ionized calcium analyzer(Orion Biomedical Instruments) to assure that it had negligiblecalcium-binding capacity. This was done both as an additional check onthe stoichiometry of Gd binding, and as a security measure to excludeany possibility of an acute decrease in serum calcium followingintravenous injection (thereby avoiding cardiovascular complications andtetany).

Two other soluble DTPA-dextran derivatives of differing molecularweights were synthesized from starting dextrans of 10,000 MW (DextranT10, Pharmacia Chemicals) and 40,000 MW (Dextran T40, PharmaciaChemicals). These reactions were carried out in a fashion analogous tothat just described, and the resulting conjugates were allowed tochelate Gd at stoichiometric quantities (determined by Gd+EDTAcomlexiometric titration against xylenol orange), to form a) solubleGd-DTPA-dextran T10 (MW=11,000; R1=4.24/(mM×sec)); and b)Gd-DTPA-dextran T40 (MW=43,000).

2. Preparation of Microaggregates From the Soluble Polymer Conjugate

If desired, microaggregates (ranging from 3 to 100 nanometers indiameter) are produced directly from the soluble Gd-DTPA-dextran T70polymer by adding NaOH to the product (at a concentration of at least 8%(w/v) in 0.02M phosphate buffer+0.15M NaCl) until the final pH is 8.2 orgreater (preferably 8.5-9.0) and incubating the product for 16-48 hoursat either room temperature or 4° C. Microaggregates form based on ioniccharge effects, and these are stable from the standpoint ofbiodistribution to reticuloendothelial organs following intravenousadministration (see Example 3).

B. Conjugation in Nonaqueous Solvent

This was carried out as above, except by suspending the initialreactants in N,N-dimethylformamide (preferred due to favorabletemperature stability). A second solvent expected to allow comparableconjugation, is N,N-diethylacetamide; this may have a biologicaladvantage comprising improved susceptibility of its two-carbon fragmentsto metabolism, and hence, reduced toxicity in vivo if trace quantitiesof organic solvent remained with the DTPA-dextran following dialysis.Because neither of the substrates (dextran nor DTPA anhydride) are fullysoluble in N,N-dimethylformamide the kinetics of conjugation are quiteslow (ca. 12 to 16 hours). Consequently, for the present reaction, 44 mgof highly pure his-cyclic DTPA anhydride (Calbiochem-Behring Corp.) wasadded to 20 mg of Dextran T70 (average MW=70,000 daltons, PharmaciaChemicals) suspended in 1.5 cc of dry N,N-dimethulformamide (BakerChemicals) and the conjugation was accelerated byultrasonification+vigorous stirring of the reaction mixture, either withor without cooling at 4° C. (both temperatures gave equivalent results).Under these conditions, DTPA conjugation proceeded rapidly and plateauedat 15 to 30 minutes. At this point, NaOH was added either a) in the formof powdered pellets at the completion of conjugation, just prior tohydrolyzing any excess unreacted DTPA dianhydride with a 2-fold excessof water (with vigorous stirring and sonification); or b) in the form ofan aqueous solution, at the same time as hydrolysis of any excessunreacted DTPA dianhydride (both methods gave equivalent results). Inattempts to minimize microaggregation due to pH, the quantity of NaOHwas carefully adjusted to give a pH of 6.0 upon formation of the aqueousmixture. This nonaqueous method resulted in a 1.7 fold increase incomplexing ratios of Gd to dextran T70 and dreased the DTPA dianhydriderequired by a factor of 1.8. As recently prepared inN,N-dimethylformamide, one of every 7.2 sugar residues is conjugated toan active DTPA ligand, for a total of 54 Gd-binding ligands per 389glucose units. The resulting product has an R1 in physiologic saline ofgreater than 50/(mM×sec). As assessed a) in vitro by microscopy andlight scattering, and b) in vivo by biodistribution patterns (see below)the physical form of this organic-phase, DTPA-dextran conjugate wasmoderately to heavily predominantly microaggregated, with sizes rangingup to 0.1 to 0.2 micrometers in diameter.

An analogous, organic-phase synthesis was carried out by dissolvingseparately, the identical quantities as above of DTPA anhydride anddextran T70 in dimethylsulfoxide, mixing these together after maximalsolution of the individual reagents had occurred, stirring the mixturefor 8 to 18 hours at either 4° C. or 22° C. (both methods producedequivalent results, and hydrating the conjugates slowly (while stirring)with distilled water, with maintenance of pH at 7.0. Both methodsproduced conjugates whose physical form was moderately to heavilymicroaggregated, with sizes ranging up to 0.1 to 0.2 micrometers indiameter (assessed in vitro as described just above).

Compared to the preceding, aqueous-phase method, the slight advantagesin substrate savings and derivatization ratios observed with thisorganic-solvent synthesis, are markedly offset by: a) the intermolecularcross-linking (covalent microaggregation) which occurs in (and istypical of) organic-phase conjugation (due to the very slow rate ofsecond-group anhydride hydrolysis in nonprotonating solvents); and b)the reduced Gd relaxivity which apparently results frommicroaggregate-induced impedance of water proton exchange rates (seeExample 4, table). This in turn, leads to marked disadvantages in theproduct's biodistribution, clearance rates, and tumor access followingintravenous administration (see Example 3). Hence, aqueous- rather thanorganic-phase conjugations are the preferred methods for synthesizingpolymeric intravascular contrast agents because organic-phase synthesisdoes not allow the formulation of completely soluble (noncross-linked)products. This feature is imperative for medical utility and regulatoryacceptance.

C. General Features of DTPA-dextrans

Dextran-DTPA image-enhancing agents particularly with entrainedgadolinium, were produced under a variety of conditions and withdifferent dextrans in various batches. Each batch was lyophilized and,when stored at room temperature, found to be stable at 22° C. in excessof 1 year. Physiologic solutions of these agents were equally stable andgave no release of free Gd after 1 year at 4° C. Particular batches ofdextran-DTPA image-enhancing agents were prepared having molecularweights of 10,000, 40,000 and 70,000 daltons although the method isusable for a size range of at least from 1,000 daltons to 2,000,000daltons. The high derivatization ratio, which was observed for bothaqueous and nonaqueous conjugations results from the increased ease offorming ester bonds relative to the amide bonds formed in conjugationsto primary amines of proteins (see Background). Whereas ester bonds aresufficiently stable to allow initial targeting and tissue and cellularuptake which parallels that of the carrier molecule, these bonds arealso more rapidly biodegraded in host cells add serum than are peptidebonds. This decreases toxicity by allowing faster cleavage of Gd-DTPAfrom the localized carrier, and hence, faster release from these sitesof initial localization (entrapment) and more rapid and completeclearance of Gd-DTPA from the body by renal excretion (see Example 3).The increased rotational correlation time of the dextran macromoleculeand its hydrophilic nature (which allows rapid on-off binding of waterprotons) amplify the paramagnetic efficiency (specific activity) of eachGd by multiples of 4.5 for the aqueous conjugate (soluble) and 2.2 forthe nonaqueous conjugate (microaggregates) (see Example 4, Table). Thenet negative charge of hydroxyl Groups on the glucose residues (whichare slightly ionized at physiologic pH) contributes to stabilization ofGd⁺³ binding by electrostatic effects and hence increases the Gdstability constant to significantly above 10¹⁷. The combination of theseproperties cause the dose, in vivo bioexchange and toxicity of Gd to besubstantially decreased. The high derivatization ratio (Gd-DTPA perdextran) also minimizes the amount of carrier material required for MRimage enhancement in vivo. This reduces the total osmolality to levelswhich allow acute intravenous injection of MRI doses without producingunacceptable acute plasma volume expansion.

EXAMPLE 3 IN VIVO PHARMACOKINETICS AND BIODISTRIBUTION OF THEIMAGE-ENHANCING AGENTS A. Soluble Agents

The soluble, 78,000 MW dextran-DTPA gadolinium chelates described inExample 1 (aqueous solvent method) have been injected directly into miceand rats. At the usual doses of 25 to 250 mg/kg, in rats, the chelatedGd has a blood clearance whose two major components have t1/2's of about50 and 180 minutes, as assessed by radioisotopic 153Gd. This provides upto a3-fold increase in the MR imaging window compared to Gd-DTPA. Theflexibility exists for coupling DTPA to biocompatible carbohydratecarriers of various molecular weights, ranging from 1,000 to 2,000,000daltons. By using shorter chain lengths than 70,000 daltons (e.g. 1,500to 40,000 daltons) clearance times could be shortened towards those ofGd-DTPA. This may also increase slightly the propensity of the contrastmaterial to extravasate into tumors and inflammatory lesions.Alternative mono-, di-, oligo- and polysacchardes potentially includealpha, beta and gamma cyclodextrins, poly-cyclodextrins, glucose,glycogen, maltose, starch (and its derivatives, e.g., hydroxyethyl,carboxymethyl-, and aminoethyl-) blood-group oligosaccharides and theirderivative amines, mucopolysaccharides and their oligomers, heparins,heparan, heparan-SO₄, chondroitin-SO₄, dermatan-SO₄, and related,natural and synthetic, water-soluble polycarbohydrates and theirderivatives.

In mice, the blood clearance of the Gd in ¹⁵³ Gd-DTPA-dextran 70 occursin 1/2 to 1/3 the (t1/2) time observed in rats (above). Whereasclearance in rats is more predictive of that in humans, this acceleratedclearance in mice has important implications for several of thesubsequent examples involving in vivo potencies (in both theT1-relaxation and MR imaging modes), as follows. First, comparison ofthese NMR changes at a fixed time interval (e.g. 30 minutespost-injection) will make the soluble polymer appear to be more potent(for tumor imaging) in rats than in mice, whereas, if compared at timesof equal blood levels, these two species of animal give equal results.Second, when the soluble polymer is compared with its microsphereformulation (made from the same soluble polymer) at e.g., a 30-minutepost-injection interval, the microsphere formulation will appear to beconsiderably more potent (at enhancing liver) than is the solublepolymer (at enhancing tumors). This is because microsphere clearancefrom the liver occurs an order of magnitude more slowly (see below) thandoes soluble polymer clearance from a typical tumor. However, whenmonitored at appropriately shorter post-injection intervals (20 minutesin mice; 30-45 minutes in rats) the soluble polymer is actually verysimilar in potency to microspheres.

B. Microsphere Agents

In both rats and mice, the t1/2 for blood clearance of the Gd inGd-DTPA-dextran microspheres (0.1 to 0.5 micrometers in diameter) is ca.15-20 minutes (as assessed by NMR T1 changes in the freshly excisedorgans). In rats, ca. 50% of this microsphere Gd is cleared within ca. 2hours by the kidneys (same method). Initial studies (using bothradioisotopic ¹⁵³ Gd and NMR T1 methods) indicate that the residualfraction of microsphere Gd which remains entrapped in the liver beyond 2hours, clears with a t1/2 of 5-6 days. This slower clearance occurs byboth the gastrointestinal (major) and renal (minor) routes and iscomparable in rate to that for the liver clearance of native dextran 70.

C. Microaggregated Agents

In mice, the blood clearance of the Gd in Gd-DTPA-dextranmicroaggregates (3-100 nanometers in diameter) ranged from 60-240minutes depending of their size (t1/2's increasing with smallersize--¹⁵³ Gd method). Biodistributions also varied depending on size,however, typically 40% to 75% of the agent was cleared by the liver.Maximal liver levels occurred at 24 hours post injection. Subsequentliver clearance occurred with a t1/2 of 5-6 days (same method).

Comparative Biodistributions of Soluble and MicroaggregatedGd-DTPA-dextrans

The following table illustrates typical biodistribution results obtainedat 33 minutes post-injection of ¹⁵³ Gd-DTPA-dextran T70 in tracer doses,as the soluble polymer and microaggregates, into Swiss nude mice bearinghuman (BRO) melanoma tumors.

    ______________________________________                                        Organ concentration of Gd (uM)                                                             Soluble  Microaggregated                                         Organ        Agent    Agent                                                   ______________________________________                                        Liver        3.3      142.0*                                                  Tumor        6.8      3.8                                                     (maximal)                                                                     Kidney       18.7     16.2                                                    ______________________________________                                         *Liver sequestration of these microaggregates continues to increase over      time and peaks at ca. 24 hours at 150% of this 33 min value. (This delaye     increase is not observed for the soluble agent.)                         

For the microaggregated agent, tumor concentrations were reducedabsolutely due to: a) strong competitive uptake by the liver, and b) theinaccessibility of supramolecular aggregates to tumors because of thesmaller size of tumor capillary "pores".

EXAMPLE 4 PRODUCTION AND USE OF MICROSPHERES

The soluble polymer of Example 2 has also been reformulated as verysmall (0.1-0.5 um) hydrophilic microspheres, by a modification of themethod reported by the Applicant in a recent issue of Science (V 227, p182 (1985)). In summary, this method involved first the emulsificationof the dextran-DTPA-Gd complex in an oil such as cottonseed oil. Theemulsified complex was then sonicated to produce smaller microspheres.The oil was extracted with a volatile organic solvent (ether or hexanes)and the microspheres were lyophilized. In contrast to the liposomes andcolloids discussed earlier herein, these new, very small hydrophilicmicrospheres allow almost complete access and rapid exchange of waterprotons to all the Gd throughout the sphere matrix. The R1 ofmicrospheres in physiologic saline is greater than 90/(mM×sec). Hence,microsphere-Gd and polymer-Gd have almost identical T1 activities invitro. This is consistent with the reported finding that increments inGd relaxivity, which are produced by macromolecular coupling, plateau atmacromolecular weights ≧65,000 daltons (Lauffer et al. (1985) Mag. Res.Imaging V 3, p 11). Hence, the slower rotation of microspheres relativeto the soluble polymer, is not expected to give any further improvementin the relaxivity of microsphere-Gd over soluble macromolecular Gd(except potentially under flow conditions--see Example 8, below.

On intravenous injection, the microspheres are cleared (capturedinitially) spontaneously by the liver, spleen and bone marrow of miceand rats (at a t1/2 of approximately 15 minutes). Here, they undergocontrolled dissolution to the soluble polymer at a t1/2 of 30 minutes.This selectively enhances NMR images of the preceding organs. Optimal T1decreases have been obtained in the livers of mice using lower injecteddoses of Gd (0.01 to 0.02 mmoles/kg) than are normally used for standardcontrast enhancement in clinical imaging (Gd-DTPA, 0.10 to 0.30mmoles/kg). The latter agent produces minimal changes in liver Tl's atthe usual 30-minute imaging interval.

In rat studies, enhancement of liver images is achieved with microspheredoses 10 to 27 times lower than those required for Gd-DTPA. Thissignificant dose advantage is produced by the combined effects of fourdesign features: the increased rotational correlation time ofmicrosphere-Gd, the improved permeation of water protons into thehydrophilic matrix and rapid on-off binding to (or near) Gd, theextremely small diameters of the microspheres, and the selective uptakeof microspheres by target organs. As a result, these microspheres areeffective in vivo at the lowest doses of any formulation reported (downto 0.007 mmoles/kg).

The following table describes many of the above described results.

    ______________________________________                                        CONCENTRATIONS OF TOTAL MATERIAL AND                                          GADOLINIUM REQUIRED TO PRODUCE A 50%                                          DECREASE IN THE T1 OF WATER PROTONS (IN VITRO)                                (IBM PC 20 MINISPECTROMETER, 20 MHz)                                          Gado-                                                                         MaterialliniumGd                                                              Concen-Concen-specific                                                        trationtrationactivity                                                        Sample Material(ug/ml)(m/10.sup.-5)ratio*                                     ______________________________________                                         ##STR1##                                                                      ##STR2##                                                                     Dextran-DTPA-Gd                                                               synthesized in DMF organic                                                    solvent (1 DPTA/7                                                             glucose) 804.35                                                               ______________________________________                                         *Lower gadolinium concentrations indicate a higher .sup. 1 H relaxivity       per Gd atom. A higher specific activity ratio for T1 corresponds to an        increase in the MR signal intensity achieved per gadolinium atom (and an      increased image intensity obtained in vivo).                                  **These lines indicate the relationships producing the ratio.            

EXAMPLE 5 IN VIVO NMR ENHANCEMENT OF NORMAL TISSUES: LIVER, SPLEEN ANDBONE MARROW

Sprague-Dawley rats were imaged using a 0.35-Tesla, Diasonics clinicalMR imaging system and a 30-cm rf coil. Three clinically relevant pulsesequences were used: 1) spin-echo with a TR of 0.5 seconds (forT1-weighted images), 2) inversion-recovery (IR) (for T1-weightedimages), and 3) spin-echo with a TR of 2.0 seconds (for T2-weightedimages). Diasonics software was used to calculate the area-averagedtissue intensities before and after injection of contrast agents. Dualpulse sequences (spin-echo, with TR's of 0.5 and 1.5 seconds or 1.0 and2.0 seconds) were also used to calculate the in vivo T1 relaxationtimes. At the conclusion of imaging, the liver, spleen and kidneys wereexcised and their T1 (IR) and T2 (Carr-Purcell-Meiboom-Gill) relaxationtimes were determined at 37° C., using an IBM PC20 Minispectrometer. Forthese in vitro experiments, uninjected rats were used in place ofpreinjection controls.

Two contrast materials were compared at equivalent in vitro doses: 1)Gd:DTPA dimeglumine (0.3 mmoles/kg; Schering AG), and 2) the 0.1-0.5 umhydrophilic Gd:DTPA-dextran microspheres prepared by the Applicant fromNo. 2 (0.009 mmoles/kG); with an in vitro potency 4.1 times that ofGd:DTPA dimeglumine). Post-contrast images were obtained seriallybeginning immediately after the i.v. injection of contrast agents andcontinuing for several hours thereafter. At 30 minutes post-injection,the three enhancing agents decreased image T1 values as follows:

    ______________________________________                                        % Decrease in T1                                                              (30 min post vs. pre)                                                                           liver                                                                              kidney                                                 ______________________________________                                        Gd:DTPA dimeglumine:                                                                              24.6   55.7                                               Gd:DTPA-dextran     54.5   26.0                                               microspheres                                                                  ______________________________________                                    

Image intensities were increased (enhanced) in inverse proportion to thedecreases in T1 relaxation times. The organ pattern of T1 changes (Tableabove) documented selective liver uptake of Gd:microspheres but notGd:DTPA dimeglumine (which was concentrated instead, in the kidney).Liver enhancement by Gd:microspheres persisted unchanged for 2.5 hoursafter Gd:DTPA microspheres but not after Gd:DTPA dimeglumine. (For thislatter agent, all liver enhancement was lost after 50 minutes). Thus,optimal selective enhancement of liver was achieved by Gd:DTPA-dextranmicrospheres at a Gd dose 10-20 times lower than that required forGd:DTPA dimeglumine agent. These Gd-microspheres also prolonged theinterval of image enhancement from minutes to hours. In thepost-injection images of animals which received Gd-microspheres (but notGd:DTPA dimeglumine), the image pixels corresponding to bone marrow werecomparably enhanced as assessed by visual inspection, however, thenumber of pixels corresponding to each rat bone was too small tonumerically quantify these changes. Rat spleens could not be imaged dueto their small size and proximity to liver, however, in vitro T1 changesof the freshly excised organs indicated that the spleens had enhanced T1's in proportion to those of liver (see next paragraph).Pre-administration T1's were compared to T1's from spleens 35 min afteradministration of the agents.

T1 and T2 relaxation times of freshly excised organs (read in an IBMPC20 Minispectrometer) decreased in proportion to those obtained fromthe imager. T1 changes uniformly exceeded the changes in T2 times. Inparticular, the normalized in vitro T1 changes in rat spleens were:

    ______________________________________                                        % Decrease in T1 of Spleen                                                    (30 min post vs. pre)                                                         ______________________________________                                        1.         Gd:DTPA dimeglumine:                                                                          14.4                                               2.         Gd:DTPA-dextran 61.2                                                          microspheres                                                       ______________________________________                                    

Hence, combined in vivo and in vitro analyses indicated thatGd:DTPA-dextran microspheres gave markedly improved enhancement of MRimages and/or T1 relaxation in the predicted target organs=liver, bonemarrow and spleen.

EXAMPLE 6 IN VIVO NMR IMAGE ENHANCEMENT OF A PRIMARY LIVER TUMOR(HEPATOMA)

Using a direct needle-puncture technique, cell suspensions of the7777-strain, syngeneic, transplantable, metastasizing Morris hepatomawere injected orthotopically into the right lobes of the livers in 650gm, Buffalo-strain rats. After two to three weeks the local tumors hadreached an average diameter of 0.5 and 1.0 cm. The rats were then imagedboth before and after i.v. injections of Gd-DTPA or microsphere Gd-DTPA.MR imaging was performed in a 30cm rf coil with a 0.35 Tesla, Diasonicsclinical MRI system (as described above). Post-contrast images wereobtained serially beginning immediately after injection and continuingfor several hours thereafter.

The Gd:DTPA-dextran microspheres produced a selective enhancement of thetumor (by visual inspection) in relation to surrounding normal liver andall other organs of the rat. Tumor enhancement was maximal in the T1modes (spin-echo with TR's of 0.5 and 1.0 sec; and inversion recovery)but was also observed in the T2 mode (spin-echo with TR of 2.0 sec).Tumor enhancement became strong at 25 minutes post-injection andpersisted unchanged over the 2.5 hour interval of post-injectionimaging. Gd:DTPA-dextran microspheres (at 0.011 mmoles/kg) producedimage enhancement comparable in intensity to that of Gd:DTPA dimeglumine(at 0.1 mmoles/kg).

The major differences between these two agents were dose(Gd-microspheres gave a more homogeneous enhancement with improveddemarcation (contrast) between tumor margins and adjacent normal liver),and persistence of contrast (Gd=DTPA-dimeglumine contrast wassignificantly reduced by 1.5 hours after injection). In vivoquantification of the increase in tumor image intensity was difficult toobtain because of the small volume of tumor tissue and tumorinhomogeneity. However, in vitro T1 measurement performed on the excisedtumor and liver at 2.5 hours corroborated the overall tumor enhancementobserved in vivo, as follows:

    ______________________________________                                        Buffalo Rat Tissues in Vitro                                                  T1 (milliseconds)                                                                     Tumor (hepatoma)                                                                          Adjacent normal liver                                     ______________________________________                                        Pre-injection                                                                           782           330                                                   Post-injection                                                                          530           320                                                   (2.5 hours)                                                                   ______________________________________                                    

The percentage decrease in T2 relaxation of tumor tissue post-injectionwas approximately 2/3 of that observed for T1. The result of enhancementwas to brighten the tumor image in relation to surrounding normal liverand other abdominal organs.

EXAMPLE 7 IN VITRO NMR T1 RELAXATION ASSESSMENT OF A NON-LIVER TUMOR(RIF SARCOMA) FOLLOWING IN VIVO INJECTION OF GD:DTPA-DEXTRANMICROSPHERES

The selective uptake by a primary liver tumor (hepatoma) ofGd-microspheres at the expense of uptake by surrounding normal liver andother body organs was unexpected but reproducible for the 7777 hepatomaline. This fortuitous result was suspected to be atypical for most soldtumors, due to the general absence in such tumors of phagocytic cellsresponsible for microsphere-Gd uptake. This was further tested byinjecting C3H mice in the legs with syngeneic, transplantable RIFsarcomas, allowing the tumors to grow to 1 cm in diameter, and theninjecting the mice with Gd:DTPA-dextran microspheres i.v. at a dosecomparable to that used above. Pre- and post-injection (45-min) tumors,livers and kidneys were excised and tested in the IBM PC20Minispectrometer for effects of Gd-microspheres on T1 relaxation times.The results were as shown below.

    ______________________________________                                                      T1 of Control                                                                            % Decrease                                           Organ/Tissue  (msec)     post-injection                                       ______________________________________                                        Tumor         804        3.9                                                  Liver         370        20.5                                                 Kidneys       434        7.9                                                  ______________________________________                                    

Although these latter tumors were in their orthotopic rather thanhepatic (liver) locations, the results still suggest strongly that forthe usual case of non-primary liver tumors which invade the liver, tumortissue will, as anticipated, selectively exclude microsphere-Gd, and thesurrounding normal liver will relatively concentrate the microsphereagent, leading to enhanced contrast in the reverse pattern from thatobserved for the preceding hepatoma, namely brighted normal liversurrounded by relatively darker tumor nodules.

Advantages of Gd:DTPA-dextran microspheres as an enhancing agent forliver lesions (and also for spleen and bone marrow lesions) include:

1. Detection of lesions at smaller (potentially millimeter) sizes;

2. Improved demarcation of tumor margins for evaluation of surgicalrespectability;

3. Prolonged enhancement interval (of hours) for performing serial MRimages and shortening the time required for each image;

4. Administration of the lowest dose of Gd (0.007 to 0.024 mmoles/kg)resulting in production of the most minimal toxicity possible with aliver-specific paramagnetic enhancing agent.

EXAMPLE 8 VITRO NMR T1 RELAXATION ASSESSMENT OF A NON-LIVER TUMOR (RIFSARCOMA) FOLLOWING IN VIVO INJECTION OF TWO GD:DTPA-DEXTRAN SOLUBLEPOLYMERS

C3H mice, bearing 1 cm transplantable, syngenic RIF sarcomas in theirlegs (see Examples above), were injected i.v. with two soluble polymericforms of Gd:DTPA-dextran at a Gd dose of 0.09 mmoles/kg. Tumors, liversand kidneys were excised from pre- and post-injection animals at 60-75min after injection, and the T1 relaxation times of organs and tumorwere determined in the IBM PC20 Minispectrometer for the effects oflocalized Gd.

    ______________________________________                                                    MW of                                                                         Polymer  T1 of Control                                                                              % Decrease                                  Organ/Tissue                                                                              (/1000)  (msec)       post-injection                              ______________________________________                                        1. Tumor    70       804          15.7                                                    10       "             3.2                                        2. Liver    70       370           0.6                                                    10       "             7.7                                        3. Kidneys  70       434          39.7                                                    10       "            21.2                                        ______________________________________                                    

These results indicate that, as predicted for a nonprimary liver tumorsuch as RIF, the larger (70,000 MW) soluble polymeric form ofGd:DTPA-dextran gives the reverse pattern of uptake by tumor and liverrelative to that just documented for the Gd-microsphere formulation.(This pattern is not seen with the 10,000 MW polymer due to itsrelatively rapid renal clearance (see above table). If the RIF tumorwere Grown in the liver rather than leg of mice, selective uptake of the70,000 soluble Gd-dextran polymer by intrahepatic RIF tumor would beexpected to produce image brightening in the tumor and an unchangedimage intensity in the surrounding normal liver (a pattern of enhancedimage contrast parallel to that shown above for Gd-microsphereenhancement of liver hepatoma).

EXAMPLE 9 ENHANCED NMR IMAGES OF BLOOD BASED ON CARDIAC DIFFERENTIALFLOW WITHIN CHAMBERS

Studies were performed indicating that intravenously administeredmicrospheres enhanced T1-weighted blood flow images in the chambers ofrat hearts (ungated, 5 minute images), at times up to 20 minutes afterinjection. Gd:DTPA-dextran microspheres (at 0.3 mmoles Gd/kg) wereinjected i.v. at time zero into Sprague-Dawley rats and images wereobtained immediately and serially each 5 minutes×4 (spin-echo,multi-echo, TR=0.5 and 1.5). Under normal flow conditions, imageenhancement was most prominent in the regions of slower flowing bloodadjacent to the endocardial surfaces. However, under conditions ofgeneralized slow flow (induced by co-injecting a polycationic polymer attime 0), all portions of the cardiac chambers gave enhanced T-1 weightedblood images.

The soluble Gd:DTPA-dextran polymer, injected at a comparable Gd dose,produced analogous but slightly weaker enhancements. The superiorperformance of microspheres under flow conditions suggests that factorsrelated to flow turbulence are more effectively overcome by particlesthat by molecular carriers, and by larger molecules than smaller ones.This interpretation is supported by the finding that the very small MWenhancing agent, Gd-DTPA (dimeglumine) was almost completelyineffective. This ineffectiveness held true even when injections weremade directly into the heart and imaged immediately with cardiac gating(R. Peshock, unpublished studies). Hence, it appears that the two newcontrast agents are the only ones potent enough to produce noninvasiveenhancement of blood flow images with the available methods of clinicalMR cardiac imaging.

EXAMPLE 10 TOXICOLOGY A. Soluble Gd-DTPA-dextran T70

The LD50 in CBA mice of intravenously injected Gd-DTPA-dextran T70 was12.3 gm/Kg. This was identical to the LD50 of native dextran andresulted from the acute osmotic effects of the dextran carrier. For thepreparation used, this total dose corresponded to 2.26 mmol of Gd perKg. The ratio of toxic/effective dose was 90 (=2.26/0.025 mmol of Gd perKg). Also, the LD₅₀ of Gd-DTPA-dextran T70 was 5 times higher than theLD₅₀ of GdCl₃. This indicated that there was negligible in vivo release(bioexchange) of chelated Gd.

No significant acute or subacute hepatotoxicity or renal toxicity wasobserved by histologic assessment after administration of 5 times theeffective MRI dose. All mice remained normally active, ate and dranknormal quantities of water, and gained weight at the same rate asuninjected litter mates.

B. Microsphere Gd-DTPA-dextran T70

In toxicologic tests, the LD₂₀ of Gd-DTPA-dextran microsphereswas >1,250 mg/kg. To put this in perspective, image enhancement iscarried out at less than 1/5th to 1/11th of the LD₂₀ dose, depending onthe preparation used. Also, histologic assessment of the major organsexcised after MR spectroscopy (in CBA mice) and MR imaging (inSprague-Dawley and Buffalo rats) revealed no evidence of acute (30-60min) toxicity.

Preliminary subacute toxicologic studies were performed on CBA mice byinjecting them at time 0 with Gd:DTPA-dextran microspheres at a dose(250 mg/kg; 0.06 mmoles/kg Gd) which was approximately 2.5 times thestandard dose used for imaging procedures. This was followed by minorelevations (less than or equal to 2 fold) of the liver enzyme, serumglutamic-oxaloacetic transaminase (SGOT) which peaked on day 3-4 at 140%of the values for upper limits of normal and fell back to nearly thecontrol range by day 7 post-injection.

Subacute histologic assessment of the liver (which was assessed by boththe Applicant and a specialist in liver pathology) revealed minor zone 1and 2 changes beginning at 6 hours post injection and comprising slightswelling and vacuolation of hepatocytes. This culminated at day 3-4 inextremely rare single-cell dropout without changes in the quantity orappearance of supporting connective-tissue or portal tracts. Thesechanges largely resolved by day seven. No significant changes in serumcreatinine (an indicator of renal function) or renal histology wereobserved over the 7-day test interval. The mice remained normallyactive, ate and drank normal quantities of water, and gained weight atapproximately the same rate as their uninjected litter mate controls

EXAMPLE 11 PREPARATION AND TESTING OF GLYCEROL-DTPA COPOLYMER

Dried glycerol (0.4 ml, 0.55 Mole) was added to DTPA cyclic dianyhydride(296 mg) suspended (by sonification) in 0.4 ml of dried,N,N-dimethylformamide. This mixed suspension was sonicated for anadditional 3 min at 20,000 Hz with a special microtip (Heat Systems,Inc.) and heated for 7 hours at 135° C. to give controlledpolymerization, plus an additional 2 hours at 155° C. to drive off thereaction solvent (BP=149°-156° C.). The resulting resin was transferredsegmentally with sonication into 60 ml of distilled water (pH 5) andsheared for 20 minutes with a high-speed Waring blender. GdCl₃.7.05H₂ O(327 mg) was adjusted to pH 5, added dropwise to the DTPA-glycerol resinand again sheared for 3 hours to maximally solubilize the material. Theresidual larger gel-state material was separated by centrifugation at250×g for 15 min, and the smaller soluble fraction was saved andseparated from residual free Gd by molecular filtration (with 4 washesof distilled water, pH 5.0) through a 1000 MW cutoff filter underpressurized nitrogen. The retentate was saved and centrifuged for anadditional 15 min at 1000×g and the supernatant of this was saved andlyophilized 16 hours.

Although initially it had the appearance of a gel, the resultingglycerol-DTPA:Gd copolymer was minimally to negligibly cross-linked asdetermined by molecular filtration, which gave a size range (for 95% ofthe material) of 1,000 to 10,000 MW, with an estimated average of 2,200MW. This confirmed that the copolymeric units were soluble but that theyhad a tendency, as formulated presently, to undergo ionic intermolecular aggregation at a high concentration, which was reversible at alow concentration.

In vitro testing for T1 relaxation effects in the IBM PC20Minispectrometer gave the following result (compare, for example thetable of Example 3):

    ______________________________________                                        Dose Decreasing T1 by 50%                                                                  total wt (ug/ml)                                                                         Gd (M/10.sup.-5)                                      ______________________________________                                        Gd:DTPA-glycerol                                                                             32           5.0                                               copolymer                                                                     ______________________________________                                    

Thus, on the basis of Gd molarity, Gd:DTPA-glycerol copolymer was 1.9times as active as Gd:DTPA dimeglumine. Its R1 was greater than 100/(mMand sec).

In vivo tests were carried out by injecting CBA mice i.v. with 130 mg/kgof Gd:DTPA-glycerol copolymer and determining the effects of T1relaxation times of organs freshly excised at 30 minutes after agentadministration.

    ______________________________________                                               Control T1                                                                              Injected T1                                                         (msec)    (msec)    % Decrease                                         ______________________________________                                        Liver    339         200       41.0                                           Kidney   343         223       35.0                                           ______________________________________                                    

Thus, as presently formulated, Gd:DTPA-glycerol copolymer wasconsiderably more active as a MR enhancing agent for liver than wasGd:DTPA dimeglumine on both a weight and Gd molar basis. (It is expectedthat this effect should be overcome by formulations which decreaseintermolecular aggregation by altering electrostatic charge or pH; or byadding inert, chain-separating molecules. Preliminary acute toxicologicstudies were very slightly inferior to those of the Gd:DTPA-dextransoluble polymer. It is anticipated that this toxicity should be improvedby substituting the hexadentate chelator, TTHA, for DTPA. This wouldleave 4 carboxylic acid groups available for Gd chelation (as withDTPA-dextran), and hence, theoretically decrease Gd bioexchange in vivo.

EXAMPLE 12 CONJUGATION OF BINDING GROUPS TO THE GD:DTPA-DEXTRAN POLYMERSAND MICROSPHERES

Approximately 50 mg of the dextran-DTPA polymer or 150 mg of particleswere suspended in 9.5 ml distilled water with 0.05M NaCl. Sodiumperiodate (0.05M, 300 ul) was added and the mixture stirred at 22° C.for 30 min. The preparation was washed with distilled water (bymolecular filtration, polymer; or centrifugation, microspheres) andbrought up in 15 ml distilled water or saline. To the periodate-oxidizedpreparation were added the materials to be covalently conjugated:antibody, avidin, or biotin hydrazide, at 1-3 mg each, depending on thenumber of reactive groups on the additive. This mixture was stirredagain for 30 minutes, then NaBH₄ (8 mg) was added to reduce the Schiffbase (or its equivalent) and stirring was continued for an additional 15min. The pH was adjusted to 7.5, and the stabilized preparation waswashed and resuspended in 1 ml of 0.02M phosphate-buffered in 0.15M NaClcontaining 0.25% dextran T70. Microspheres derivatized by this methodhad between 2500 and 5000 available binding sites per 0.5 urn sphere, asassessed by the high-stability specific binding of ¹²⁵ I-avidin tobiotinylated microspheres prepared with biotin hydrazide.

This method allows the direct covalent conjugation of antibodies andother receptor binding proteins or peptides via their reactive aminogroups; and the indirect coupling of (a) biotinylated antibodies(commercially available) to avidin-derivatized polymer or spheres; or(b) native antibodies to polymer or spheres pre-derivatized with ProteinA (Pharmacia Chemicals) which binds the Fc region of antibodies at highaffinities

EXAMPLE 13 PREPARATION AND TESTING OF ALBUMIN MICROSPHERES CONTAININGTHE ENTRAPPED, NONCOVALENTLY BOUND METAL ION-CHELATE COMPLEX, GADOLINIUMDIETHYLENETRIAMINE PENTAACETIC ACID (Gd:DTPA)

A 0.95M solution of Gd:DTPA in dimeglumine (2×N-methylglucamine) saltform (Schering AG, Germany/Berlex Laboratories, Inc., USA) was added at0.25 ml to a maximally concentrated solution of human serum albumin (125mg, Sigma Chemical Co.) in distilled water (0.25 ml). This was stirredfor 20 minutes, added dropwise to 30 ml of cottonseed oil (Sargent WelchScientific), and sheared for 20 minutes with a high-speed Waring-typeblender to produce submicron droplets (0.1-0.6 um diameter). Thisemulsion was added dropwise to a preheated (140° C.) rapidly stirring,100-ml volume of cottonseed oil, in order to heat denature (stabilize)the albumin matrix and maintain the integrity of particles andentrapment of Gd:DTPA upon subsequent suspension in injection medium.Heating at 140° C. was continued for 10 min with high-speed shearing.The emulsion was cooled to 220° C. with continued mixing. Oil wasextracted with 6×60 ml of fresh diethyl ether (containing antioxidant)(Fisher Scientific Co.), and the resulting microspheres were lyophilizedfor 16 hrs to remove residual ether. Particles ranged from 0.1-0.5 um(diameter) with a mean of 0.3 urn (monitored by light and electronmicroscopy).

Microspheres (Gd:DTPA:dimeglumine:albumin) were tested in vitro using a20 MHz pulsed Nuclear Magnetic Resonance (NMR) spectrometer, for theircapacity to reduce the T1 relaxation time of water protons inphysiologic saline solution (0.02M phosphate-buffered, 0.15M NaCl).Activity was expressed as the concentration of material required todecease the T1 relaxation time to 50% of the value forphosphate-buffered saline (ID₅₀). Microspheres were suspended at aconcentration of 1 mg/ml by brief sonification. Because albuminmicrospheres have a fast-release (surface) component of Gd:DTPA as wellas a controlled-release (interior) component, the spheres were washed,resuspended, and diluted serially for testing.

    ______________________________________                                        Material              ID.sub.50 (total weight)                                ______________________________________                                        Unwashed microsphere suspension                                                                     0.25 mg/ml                                              Fast-release supernatant                                                                            0.30 mg/ml                                              Washed microspheres   3.8 mg/ml                                               Gd:DTPA dimeglumine   0.084 mg/ml                                             ______________________________________                                    

Microspheres (Gd:DTPA:dimeglumine:albumin) were tested in vivo byinjecting them intravenously into 25 gm CBA mice (2 animals per group),allowing 30 minutes for uptake and sequestration by liver Kupffer cells,sacrificing the mice, and testing the excised organs. The acute (30-min)biodistribution was determined by injecting microspheres trace-labeledwith ¹²⁵ I-albumin. Radioisotope was quantified in a standard gammacounter.

    ______________________________________                                                    125.sub.I counts:                                                                           125.sub.I counts:                                               % of total    per gm of                                           Organ       recovered at 30 min                                                                         target organ                                        ______________________________________                                        Blood        7.2           10.8                                               Spleen       0.8           32.8                                               Liver       57.6          119.0                                               Lungs       31.5          369.1                                               Kidneys      2.9           26.2                                               Total       100.0                                                             ______________________________________                                    

The pattern of uptake by liver and spleen (with moderate acute lungsequestration) is typical of that for small (<3 um) particles.

The T1-weighted proton relaxation times of mouse livers were quantifiedby determining the whole-organ T1 relaxation time in a 20 MHz NMRspectrometer.

    ______________________________________                                                         Liver T1                                                     Injected material                                                                              (msec)   % of Control                                        ______________________________________                                        Saline (0.15 M)  332      Control                                             Albumin microspheres                                                                           314      94.5                                                (45 mg/kg, total wt;                                                          0.1 mmol/kg Gd)                                                               Gd:DTPA dimeglumine                                                                            327      98.5                                                (0.1 mmol/kg Gd)                                                              ______________________________________                                    

At equivalent doses (normalized to in vitro T1 potency), the formulationof Gd:DTPA:dimeglumine albumin microspheres was slightly more potentthan soluble Gd:DTPA dimeglumine. The high dose of albumin carrierneeded to achieve this modest T1-relaxation, makes albumin a suboptimalmatrix material for delivering Gd to the liver for applicationsspecifically involving magnetic resonance imaging and specstroscopy.This high dose was necessitated by the marked sequestration of Gd in theinterior of microspheres and the very slow release (1/2 in 8 hrs) ofGd:DTPA from spheres which are sufficiently stabilized to give effectiveliver targeting.

EXAMPLE 14 PREPARATION OF Gd:DTPA:DIETHYLAMINOETHYL DEXTRAN SOLUBLEPOLYMER AND Gd:DTPA:DIETHYLAMINOETHYL MICROSPHERES CONTAININGNONCOVALENTLY BOUND Gd:DTPA (WITH STRONG ION PAIRING BETWEEN DTPA ANDTHE DEAE SUBSTITUENTS OF DEXTRAN); AND UNLOADEDDTPA:DIETHYLAMINOETHYL-DEXTRAN MICROSPHERES (WITHOUT CHELATED Gd)

Solution 1. Diethylenetriamine pentaacetic acid, 0.72 gms (DTPA, SIgmaChemical Co.) was dissolved in 2.5 ml distilled water, the pH adjustedto 7.2 with NaOH, mixed with GdCl³.6H² O, 0.34 gms, and the solutionreadjusted to pH 7.2 and stirred for 20 min to allow complete chelationof Gd. Solution 2. Diethylaminoethyl dextran (DEAE dextran, 500,000 MWwith 1 positively charged group per 3 glucose residues, Sigma ChemicalCo.) was dissolved by warming a saturated solution of 1 gm in 2.5 ml ofdistilled water. To prepare the soluble polymeric form ofGd:DTPA:DEAE-dextran, Solutions 1 and 2 were mixed with vigorousstirring; the pH was adjusted to 7.2 and the mixture washed twice withdistilled water (20 ml per wash) to remove unbound Gd. The solublepolymer was concentrated to 50-100 mg/ml by molecular filtration underpressurized nitrogen through a 100,000 MW cutoff filter (AmiconCorporation, XM100). To prepare the microsphere form ofGd:DTPA:DEAE-dextran, DEAE dextran was stirred vigorously into 30 ml ofcottonseed oil (Sargent Welch Scientific) until an even emulsion wasproduced. To this was added 1 ml (dropwise) of Solution 1. This emulsionwas sonicated for 6 min. (with continuous magnetic stirring) using a20,000 Hz ultrasonifier with a 3 mm special microtip (Heat Systems,Inc.) to disrupt the aqueous phase into 0.2-0.4 um microdroplets.Microparticles were stabilized and water removed by heating to 120° C.for 20 min with vigorous stirring. After cooling, oil was removed with3×60 ml of fresh diethyl ether (containing antioxidant) (FisherScientific Co.), and the sample lyophilized for 16 hrs. Microspheresranged from 0.1 to 0.3 um, with a mean diameter of 0.2 um.

Unloaded DTPA:DEAE-dextran microspheres. An alternative microsphereformulation was prepared without chelated Gd (or other metal ions), bydissolving DTPA, adjusting the pH to 7.2, and mixing this with a 1-gmsolution of DEAE dextran, all prepared as described above. The aqueousphase was emulsified in cottonseed oil and processed as described above.

EXAMPLE 15 LOADING (CHELATION) BY DTPA:DEAE-DEXTRAN MICROSPHERES OF THEPARAMAGNETIC METAL IONS, Gd⁺³ AND Fe⁺³

a. Chelation of Gd⁺³. DTPA:DEAE-dextran microspheres, 100 mg, fromExample 13, were added to 280 mg of GdCl₃.6H₂ O dissolved in 2 ml ofdistilled water and stirred for 30 min. Unbound gadolinium was removedby washing twice with 20 ml of distilled water (pH 5.5) using a 300,000cutoff, 43mm diameter filter (Amicon Corporation, XM300) underpressurized nitrogen. The microspheres were removed from the filter with2 ml of distilled water and lyophilized to dryness (16 hrs).microspheres ranged from 0.1 to 0.5 um (diameter).

b. Chelation of e . DTPA: DEAE-dextran F +3 microspheres, 100 mg, fromExample 14, were added to 350 mg of FeCl₃.6H₂ O and processed as inExample 15.a. (above) to remove unbound Fe⁺³. Particles ranged from 0.15to 0.6 um in diameter.

EXAMPLE 16 IN VITRO TESTING OF SOLUBLE Gd:DTPA POLYMERS AND Gd:DTPAMICROSPHERES PREPARED IN EXAMPLES 14 AND 15

Test materials were diluted serially and assayed for proton T1relaxivities using a 20 MHz pulsed NMR spectrometer as described inExample 3. These materials contained very minor components offast-released Gd and Gd:DTPA chelate (less than 2% of the totals). Thus,it was not necessary to wash and resuspend the materials prior to NMRtesting. The ID50 concentrations were:

    ______________________________________                                        Material            ID.sub.50 (total weight)                                  ______________________________________                                        Gd:DTPA:DEAE-dextran soluble                                                                      0.125 mg/ml*                                              polymer                                                                       Gd:DTPA:DEAE-dextran                                                                              0.160 mg/ml                                               microspheres                                                                  DTPA:DEAE-dextran microspheres,                                                                   0.175 mg/ml                                               loaded subsequently with Gd                                                   Gd:DTPA dimeglumine 0.084 mg/ml*                                              ______________________________________                                         *Molar concentrations of Gd = 4.6 × 10 - 5 M for the                    Gd:DTPA:DEAEdextran soluble polymer and 9.0 × 10 - 5 M for Gd:DTPA      dimeglumine.                                                             

The soluble Gd:DTPA:DEAE-dextran polymer was 1.96 times more potent thanGd:DTPA dimeglumine. This improved relaxivity was attributable to strongnonvocalent binding of the negatively charged, DTPA moiety of Gd:DTPA tothe positively charged, DEAE substituent groups of dextran polymer. Thelarge size of this polymer (300,00 MW) resulted in a longer rotationalcorrelation time for each noncovalently bound Gd:DTPA and allowedimproved transfer of energy from water protons to paramagnetic Gd ions.

EXAMPLE 17 IN VITRO HISTOLOGIC STAINING OF Fe:DTPA MICROSPHERES ASPREPARED IN EXAMPLE 15

Fe:DTPA:DEAE-dextran microspheres were suspended at 1 mg/ml in a 70%ethanol-water solution, 10-50 uL aliquots were placed on cytologic glassslides, the microspheres were sedimented at 750×g for 12 min. in acytocentrifuge, slides were air dried, and microsphere:Fe⁺³ was stainedfor histologic analysis by the Prussian blue, acidic ferro-ferricyanideprocedure. Dark blue reaction product formed over each microsphere, asassessed by standard light microscopy. Hence, the chelated Fe⁺³, whichwas initially bound to microsphere DTPA at neutral pH, becamedissociated sufficiently by the acidic pH of the staining solution toallow histochemical detection in vitro.

EXAMPLE 18 IN VIVO TESTING OF SOLUBLE Gd:DTPA POLYMERS AND Gd:DTPAMICROSPHERES PREPARED IN EXAMPLES 15 AND 16

a. Proton T1 Relaxation Times in Mouse Organs

Test materials were injected i.v. into 25 gm CBA mice. At 30 min themice were sacrificed by decapitation (exsanguination) and the excisedlivers and kidneys assessed for changes in proton T1 relaxation times(20 MHz; IR pulse sequence). Doses of test materials were madeequivalent based on in vitro potency (ID₅₀ analysis).

    ______________________________________                                                               T1 (%                                                               Dose      of control)*                                           Material       (mmol/kg)   Liver   Kidney                                     ______________________________________                                        Gd:DTPA:DEAE dextran                                                                         0.23        81      78                                         soluble polymer                                                               Gd:DTPA:DEAE dextran                                                                         0.23        81      78                                         microspheres                                                                  Gd:DTPA dimeglumine                                                                          0.47        83      24                                         ______________________________________                                         *The T1's of control organs were 330 msec for liver and 385 msec for          kidney.                                                                  

The soluble polymeric formulation of Gd:DTPA:DEAE dextran was the mostpotent substance for liver (approximately 4 times as potent as Gd:DTPAdimeglumine, which produced a significantly greater decrease in kidney).The microsphere form of Gd:DTPA:DEAE dextran was approximately 2 timesas potent in liver as Gd:DTPA dimeglumine. Because of selective organuptake by the liver, it produced a much smaller effect in kidney.Gd:DTPA dimeglumine was relatively ineffective at decreasing the T1 ofliver even at very high doses which produced marked decreases in kidney.(The usual dose of the dimeglumine formulation used for Phase IIIclinical trials is 0.1 mmol/kg.)

b. Proton Magnetic Resonance Imaging and Correlation With the Proton T1Relaxation Times in Rats

Test materials were injected intravenously into 500 gm Sprague-Dawleyrats. At approximately 30 minutes, the rats were placed in the head coilof a 0.35 Tesla clinical imaging device (Diasonics Corp.), andT1-weighted images (using both a spin-echo pulse sequence at TR's of0.5, 1.5 and 2.0, and an inversion recovery sequence) were obtained onimage slices of 0.5 cm thickness taken through the liver and kidneys. T1relaxation times were determined from the TR=0.5 and 2.0 data usingarea-averaging of signal intensities and a proprietary software programfor calculations. At the completion of imaging, the rats were sacrificedby decapitation (exsanguination). Their livers, kidneys and spleens wereexcised, tested for in vitro correlations with the in vivo changes inproton T1 relaxation times and then placed in buffered formalin fixativefor histopathologic evaluation.

The doses of test materials were as follows:

    ______________________________________                                                               Gd (mmol/kg)                                           ______________________________________                                        Gd:DTPA:DEAE dextran soluble polymer                                                                 0.30                                                   Gd:DTPA:DEAE dextran microspheres                                                                    0.15                                                   Gd:DTPA dimeglumine    0.30                                                   ______________________________________                                                          T1 (% of control)                                           Test Material Organ     In vivo In vitro*                                     ______________________________________                                        soluble polymer                                                                             liver     65      64                                                          spleen     NT**   48                                                          kidney    77       8                                            microspheres  liver     63      53                                                          spleen    NT      43                                                          kidney    63      23                                            dimeglumine   liver     83      88                                                          spleen    NT      86                                                          kidney    57      25                                            ______________________________________                                         *Control proton T1 values were: liver, 275 msec; spleen, 464 msec;            kidneys, 492 msec.                                                            **Not tested. Splenic images could not be obtained in the rat due to very     small organ size and anatomic juxtaposition tot he liver.                

There was a direct correlation between in vivo and in vitro T1relaxation times for liver. For kidney, the correlations were sporadicdue to difficulties in (a) determining average image intensities onthese much smaller organs, and (b) obtaining sharp demarcation betweenthe differently enhancing anatomic subregions of kidney (cortex andmedulla). Image intensities increased in inverse proportion to changesin T1. Soluble polymeric Gd and microsphere Gd gave preferential imageenhancement of liver relative to kidney, as compared to the reciprocalchanges obtained for Gd:DTPA dimeglumine. After normalizing for dose andimage intensity, microsphere Gd was 5 times more potent for liverenhancement than Gd:DTPA dimeglumine, and soluble polymeric Gd was 2.5times more potent. Bone marrow was also preferentially enhanced by thesoluble polymeric and microsphere Gd. These changes were noted visuallybut could not be quantified due to the small number of image pixelscorresponding to the bones of rats.

EXAMPLE 19 HISTOLOGIC ASSESSMENT OF THE SELECTIVE UPTAKE BY LIVER ANDSPLEEN OF MICROSPHERES CONTAINING THE PARAMAGNETIC METAL ION, Fe⁺³

Fe⁺³ :DTPA:DEAE-dextran microspheres (prepared as in Example 14) wereinjected into a CBA mouse at a dose of 140 mg/kg. Thirty minutes afterinjection, the animal was sacrificed, and the liver and spleen wereexcised. The tissues were fixed in formalin and stained using thePrussion blue (acidic ferro-ferricyanide) iron staining technique, toidentify cellular locations of microsphere iron. By microscopicevaluation, 0.1-0.6 tun (diameter) heavy concentrations (3+/4+) ofiron-positive particles were present in Kupffer cells of the liver andsinusoidal macrophages of the spleen. Other parenchymal cells of theliver (hepatocytes) and spleen were negative for iron staining, as wereother organs (bone marrow was not tested). These results indicate thatthe Fe⁺³ :DTPA chelate was bound (noncovalently) to the DEAE dextranwith a sufficient affinity to survive in vivo transit through thebloodstream and become cleared acutely as intact particle-associatediron by the phagocytic cells of liver and spleen. This histologic resultdocuments that the preferential MR enhancement of liver images and thepreferential T1 changes of splenic water proton relaxation times (above)were caused by the selective uptake of paramagnetically labeledparticles by phagocytic cells in the "target" organs.

EXAMPLE 20 ACUTE TOXICOLOGIC ASSESSMENT OF Gd:DTPA:DEAE DEXTRAN SOLUBLEPOLYMER AND THE Gd:DTPA:DEAE DEXTRAN MICROSPHERES OF EXAMPLE 14

The rats of Example 17 which were injected with soluble polymeric andmicrosphere Gd noncovalently bound (ion) paired) to DEAE dextran,developed mild-to-moderate respiratory distress between 90 and 120minutes after the injection of test materials. Based on theseobservations, histologic evaluation was performed on the formalin-fixedorgans (brain, heart, lungs, liver, spleen and kidneys) from these ratsand from CBA-strain mice injected with the same material at identicaldoses. The lungs, liver and kidneys of both the rats and mice revealedslight-to-moderate acute congestion of the small blood vessels with redblood cells). Additionally, the kidneys showed moderate acute corticaledema (accumulation of protein-poor fluid). These histologic changesdocumented an acute toxic effect of the two DEAE dextran-basedformulations of Gd. The histologic changes in CBA mice were uniformlymore pronounced than those in the Sprague-Dawley rats used for magneticresonance imaging. These inter-species differences make it uncertain ifsimilar effects would occur in humans. The nature of the majorhistologic change, acute congestion, strongly suggested that themultiple, positively charged DEAE groups of the dextran matrix, hadprobably interacted with the negatively charged surfaces of cells whichline the blood vessel walls (endothelial cells) and had inducedendothelial changes that led to red cell adherence and accumulation.From the standpoint of interpreting the image enhancement and T1 changesof Example 14, it is important that the histologic changes justdescribed did not occur at the time when the images were performed (30minutes following injection of test materials) but rather, at 90 to 120minutes following injection. Hence, taken together, Examples 16 and 18establish the efficacy (but not the biological compatibility) of Gd:DTPAnoncovalently bound to polycationic carriers, as prototype formulationsfor preferential MR image enhancement of liver, spleen and bone marrow.

EXAMPLE 21 PREPARATION OF HEPARIN-STABILIZED Gd:DTPA DIMEGLUMINEMICROSPHERES

A 0.95M solution of Gd:DTPA dimeglumine (Schering Ag-Berlex) wasconcentrated by nitrogen evaporation, adjusted to pH 10, added at 1.8 mlto 100 ml of cottonseed oil, and homogenized for 15 min with a Waringhigh-speed blender to produce fine microdroplets (0.2-0.5 um). Thisemulsion was stabilized at 130° C. for 20 min with continued shearing.Heparin, 5000 units (Upjohn Co. clinical grade from beef lung) was addedto neutralize the net positive charge of the outer surfaces of theseparticles and to confer additional particle stability upon subsequentresuspension. The positive surface charge of particles (which had beenfound in Example 19 to produce acute vascular toxicity) was conferred(in the present example) by the amine moieties of N-methylglucamine(dimeglumine). The reason for coating particle surfaces with heparin wasto neutralize this positive charge and eliminate the related acutetoxicity. The oil was extracted with diethyl ether and the particleslyophilized as in Example 12. Resulting microspheres were 0.1-0.4 um indiameter. The in vitro NMR proton T1 activity was:

    ______________________________________                                        Material          ID.sub.50                                                   ______________________________________                                        Gd:DTPA:dimeglumine:                                                                            0.045 mg/ml                                                 heparin microspheres                                                          Gd:DTPA:dimeglumine                                                                             0.084 mg/ml                                                 ______________________________________                                    

Based on the molar concentration of Gd, the microsphere formulation wasapproximately twice as active as the soluble one.

EXAMPLE 22 IN VIVO TESTING OF HEPARIN-COATED Gd: DTPA: DIMEGLUMINEMICROSPHERES

Microspheres were injected intravenously into CBA mice at a dosecalculated to deliver 0.19 mmol of Gd/kg. The percentage decreases inproton T1 relaxations of the experimental versus control (uninjected)organs excised at 30 minutes were:

Liver: 6%

Kidney: 53%

Hence these microspheres were not sufficiently stabilized to remainintact long enough for clearance by the liver and spleen (requiringapproximately 15 minutes). The addition of supplementary matrixmaterials such as 70,000 MW dextran, would be expected to confer thisrequired stability.

Histologic assessment of acute vascular toxicity was performed in CBAmice as described in Example 20. No congestion nor edema were observed.

Based on the preceding examples of polymer and microsphere efficacy,stability, and toxicity, the preferred embodiments were covalentlyconjugated dextran-DTPA polymers and microspheres in conjunction withchelated Gd.

EXAMPLE 23 IN VITRO T1 EFFECTS OF Fe⁺³ CHELATED TO DTPA-DEXTRAN SOLUBLEPOLYMER

FeCl₃.6H₂ O was added in a stoichiometric quantity to DTPA-Dextran T10(11,000 MW soluble polymer) and the T1 ID₅₀ 's compared with those ofcomparably loaded Fe:DTPA and Fe:desferrioxamine (a low molecular weightiron chelator of bacterial origin.

    ______________________________________                                                            T1 (ID.sub.50)                                            Substance           Fe(M/10.sup.-5)                                           ______________________________________                                        Fe:DTPA-dextran 11,000 MW                                                                          18                                                       soluble polymer                                                               Fe:desferrioxamine  113                                                       Fe:DTPA              37                                                       ______________________________________                                    

Fe:DTPA-dextran polymer was the most potent of 3 agents tested by amultiple of 2 over Fe:DTPA

EXAMPLE 24 In Vivo Imaging of a Human BRO Melanoma (Grown in Nude Mice)With Soluble Gd-dextran T70

These studies are performed using the rapidly proliferating, amelanotic,BRO human melanoma grown to a weight of 2.0-6.0 gms in the axillary softtissues of Swiss-strain nude mice. The amelanotic nature of the tumoravoids any potential for alteration of unenhanced tissue-relaxationtimes by naturally occurring paramagnetic substances which have beendescribed for pigmented melanomas. The axillary location of tumorsallows imaging of the tumor and liver in adjacent slices. This shortensthe imaging time for each group of mice and provides maximal anatomicseparation of tumors from the higher-intensity kidneys and bladder. MRimaging is performed on a Diasonics 0.35T instrument, using a 30-cm rfcoil, and a T1-weighted spin-echo pulse sequence (TR 500, TE 40).Pentobarbital is used for animal anesthesia in order to avoid drugmodulation of image intensities. T1 relaxation were performed on thefreshly excised organs. In general, these T1's decreased in proportionto increased image intensities. Deviations from this relationship wereobserved for enhancement with Gd:DTPA dimeglumine if the intervalbetween peak image contrast and sacrifice of the animals becameunusually prolonged. The potencies of Gd-DTPA-dextran T70 and Gd-DTPA(dimeglumine) were compared by injecting both agents at the limitingdose for Gd:DTPA dimeglumine of 0.03 mmols Gd per kg. Under theselimiting conditions, enhancement of the BRO melanoma occurredprominently with Gd:DTPA-dextran T70 but was barely perceptible withGd:DTPA dimeglumine which required a dose 1/2 log higher. Image contrastwas maintained for a significantly longer post-injection interval byGd:DTPA*dextran T70 than Gd:DTPA dimeglumine. The new soluble polymericagent had the following advantages:

1. Improved Gd potency in vivo (by a factor of ≧3.3);

2. Improved contrast gradient between tumor and surrounding normaltissues, which improves the detection of body tumors;

3. Improved discrimination of internal tumor structures;

4. Presence of an early "vascular phase" of tumor enhancement, which mayimprove the detection of small and infiltrating tumors at their mostrapidly growing borders;

5. Prolongation of tumor contrast.

EXAMPLE 25 ALTERNATIVE CHEMICAL MEANS FOR CONJUGATION OF CHELATINGSUBSTANCES TO CARRIER POLYMERS

In certain instances chemical advantages, such as increased stability ofmetal ion chelation or increased flexibility of the carrier polymer, maybe achieved by using conjugation reactions other than directderivatization with dicyclic DTPA anhydride. For example, the middleacetate group of DTPA may be selectively reacted with ethylene diaminebefore decylizing the stronger-chelating carboxylic anhydrides. This maybe accomplished by conjugation in dried organic solvents such asN,N-dimethyl-formamide or N,N-diethylacetamide using standardorganic-soluble carbodiimide techniques.

The amine-derivatized DTPA could then be reacted in aqueous solvents,using water-soluble carbodiimide reagents, with the OH groups of nativedextran, the aldehyde groups of sodium periodate-oxidized dextran (morereactive), or the carboxylic acid groups of succinylated dextran (mostreactive) which had been prepared by prior reaction with succinicanhydride. Alternatively the simple DTPA chelate could be stabilized inits most favored chelation state by prebinding Gd, followed byconjugation to ethylene diamine in aqueous solvents using water-solublecarbodiimide. Such metal-protection techniques are common methods forprotecting enzyme active sites during enzyme chemicalreactions/purifications. The resulting dextran conjugate might have evenhigher binding stability for Gd and other paramagnetic metals than doesthe completely acceptable conjugate described as the preferredembodiment in the present application.

Additional alternative methods for potentially improved or diversifiedconjugation includes (1) modified acid-catalyzed di-anhydride-alcoholreactions (W. C. Eckelman, et al., J. Pharm. Sci. (1975), 643:704); (2)amide coupling linkages between ethylenediamine derivatized DTPA andsuccinylated dextran as modified from (D. J. Hnatowich et al., J. Nuc.Med. (1981) 22:810) or direct coupling techniques involvingpentatriethylammonium DTPA, isobutylchloroformate and triethylaminehydrochloride precipitation to form the reactive species,carboxycarbonic anhydride of DTPA, which may be used for variousalternative conjugations (G. E. Krejcarek and K. L. Tucker, Biochem.Biophys. Res. Comm. (1977), 77:581). These reactions are expected toexpand and potentially improve the already satisfactory techniques ofthe present application.

In the above-described studies, radionuclide quantification of Gdbinding to the DTPA-dextran soluble polymer was performed using ¹⁵³ Gd,in collaboration with Padmaker Kulkarni, Ph.D. (Radiology, ImagingCenter, University of Texas Health Science Center, Dallas, (UTHSCD); andmagnetic resonance imaging was performed on the University's Diasonics0.35T clinical magent in a 30-cm rf head coil using T1-weighted,spin-echo and inversion-recovery pulse sequences, in collaboration withJeffrey Weinreb, M.D., William Erdman, M.D., and Jesse Cohen, M.D.,Nuclear Magnetic Resonance Imaging Center-Radiology, University of TexasHealth Sciences Center, Dallas, Tex.

Changes may be made in the construction, operation and arrangement ofthe various parts, elements, steps and procedures described hereinwithout departing from the concept and scope of the invention as definedin the following claims.

I claim:
 1. An image-enhancing agent or spectral enhancing agentcomprising:a biodegradable, water-soluble polysaccharide oroligosaccharide carrier-material comprising repeating hydrophilicmonomeric units having amino, quaternary ammonium, sulfate, carboxyl orhydroxyl groups; and a chelating agent comprising functional groupsbound noncovalently by strong ionic interaction to an amino, quaternaryammonium, sulfate, hydroxyl or carboxyl group of the monomeric units,said chelating agent having a formation constant for divalent ortrivalent metal cations at physiological temperature and pH, of at leastabout 10⁸ ; and a paramagnetic metal ion bound to the chelating agent,usable to enhance internal images or spectra, arising from inducedmagnetic resonance signals; wherein the image-enhancing agent has amolecular diameter of less than about 3 nanometers, contains less thanabout 5% (w/w) cross-linked or microaggregated species and isbiodegradable by hydrolytic or other enzymatic or physiologic mechanismsof animals or intestinal microorganisms, to intermediary metabolites,excretable chelates, polymers, oligomers, monomers or combinationsthereof, all of low toxicity.
 2. The image-enhancing agent of claim 1wherein the chelating agent is DTPA.
 3. The image-enhancing agent ofclaim 1 wherein the carrier-material has a molecular weight betweenabout 1,000 daltons and about 500,000 daltons.
 4. The image-enhancingagent of claim 1 wherein the carrier-material is dextran.
 5. Theimage-enhancing agent of claim 1 wherein the carrier-material has amolecular weight between about 40,000 daltons and about 75,000 daltons.6. The image-enhancing agent of claim 1 wherein the functional groupsare carboxyl groups.
 7. The image-enhancing agent of claim 1 in whichthe functional groups are non-covalently bound to the carrier-materialby a quaternary ammonium group covalently conjugated to thecarrier-material.
 8. The image-enhancing agent of claim 1 in which thefunctional groups are non-covalently bound to the carrier-material by asulfate group which is covalently conjugated to the carrier-material. 9.The image-enhancing agent of claim 6 wherein the chelating agent isDTPA.
 10. The image-enhancing agent of claim 7 wherein the chelatingagent is DTPA.
 11. The image-enhancing agent of claim 8 wherein thechelating agent is DTPA.
 12. The image-enhancing agent of claim 1wherein the paramagnetic metal ion is selected from the group ofelements having atomic numbers 21-29 and 57-70.
 13. The image-enhancingagent of claim 1 wherein the paramagnetic metal ion is that ofgadolinium, iron, nickel, copper, erbium, europium dysprosium, holmium,chromium or manganese.
 14. The image-enhancing agent of claim 1 whereinthe chelating agent is in a molar ratio of chelating agent/monomericunit between about 1/5 and about 1/25.
 15. The image-enhancing agent ofclaim 1 wherein the paramagnetic metal ion is that of gadolinium. 16.The image-enhancing agent of claim 1 wherein the chelating agent is DTPAand the paramagnetic metal ion is that of gadolinium.
 17. Theimage-enhancing agent of claim 1 wherein the carrier-material isdextran, the paramagnetic metal ion is that of gadolinium and thechelating agent is DTPA.
 18. The image-enhancing agent of claim 1wherein the water-soluble carrier-material comprises heparin, heparansulfate, chondroitin sulfate, dermatan sulfate, starch,carboxymethylstarch or hydroxyethylstarch individually, in combinationswith each other or with DEAE dextran.
 19. The image-enhancing agent ofclaim 18 wherein the water soluble carrier-material comprises heparinand DEAE dextran.
 20. The image-enhancing agent of claim 18 wherein thechelating agent is DTPA.
 21. The image-enhancing agent of claim 18defined further as comprising gadolinium.
 22. The image-enhancing agentof claim 18 wherein the water soluble carrier-material comprisesDEAE-dextran and heparin and the image-enhancing agent further comprisesDTPA bound to the DEAE dextran by ion-pair bonding.
 23. Theimage-enhancing agent of claim 1 wherein the water solublecarrier-material is heparin.
 24. The image-enhancing agent or spectralenhancing agent useful with external or internal magnetic resonanceimaging or spectroscopy, the agent comprising a biodegradablepolysaccharide or oligosaccharide carrier-material; a DTPA chelatingagent non-covalently bound by strong ionic interactions to said carriermaterial; and a paramagnetic metal ion bound to said chelating agent.25. The image-enhancing agent of claim 24 wherein the paramagnetic metalion is gadolinium.
 26. The image-enhancing agent of claim 25 in whichthe agent is in a completely water soluble form having a moleculardiameter less than about 3 nanometers and is useful for imagingof:internal tumors, at a potency multiple of at least about 3.3 timesthat of Gd-DTPA when administered by an intravenous route; and bodycavities and gastrointestinal tract upon direct introduction, with theexception of the stomach, due to dechelation of gadolinium at pH's ofabout 1 to 3.5.
 27. The image-enhancing agent of claim 25 in which theagent is in a completely water soluble form of less than about 3nanometers in molecular diameter, and clears rapidly from the blood andbody, primarily by a renal route, with at least about 80% of an injectedgadolinium dose clearing from the body within less than about 6 hours ofinjection.
 28. The image-enhancing agent of claim 24 wherein the agentis in a completely water-soluble form having a molecular diameter ofless than about 3 nanometers and an osmotic activity of less than about50 mOsm per kg of agent and also less than about 28 mOsm per micromoleof gadolinium.